Peptides and combination of peptides as targets or active ingredients for use in immunotherapy against AML and other cancers

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/479,552, filed 4 May 2017, which claims the benefit of U.S.Provisional Application Ser. No. 62/319,141, filed 6 Apr. 2016, andGreat Britain Application No. 1605872.9, filed 6 Apr. 2016, the contentof each of these applications is herein incorporated by reference intheir entirety.

This application also is related to PCT/EP2017/058083 filed 5 Apr. 2017,the content of which is incorporated herein by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2332.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_2912919-067003_ST25.txt” createdon 9 May 2019, and 31,174 bytes in size) is submitted concurrently withthe instant application, and the entire contents of the Sequence Listingare incorporated herein by reference.

FIELD

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I molecules of human tumor cellsthat can be used in vaccine compositions for eliciting anti-tumor immuneresponses, or as targets for the development ofpharmaceutically/immunologically active compounds and cells.

BACKGROUND OF THE INVENTION

Acute myelogenous leukemia/acute myeloid leukemia (AML) is amyeloproliferative disease that is characterized by an elevated count ofimmature myeloid blasts in bone marrow and peripheral blood. It is themost common type of leukemia in adults and accounts for approximately25% of all leukemias in the Western World. The incidence is highest inthe US, Australia and Western Europe, with about 3.8 cases per 100,000in the US and Europe (Deschler and Lubbert, 2006; Showel and Levis,2014).

AML is a disease of the elderly: The incidence for adults over 60 yearsis 15 cases per 100,000 (Showel and Levis, 2014). Patients newlydiagnosed with AML have a median age of 67 years (American CancerSociety, 2015). Males have a slightly higher risk for developing AML inmost countries (Deschler and Lubbert, 2006).

AML has the lowest survival rate of all leukemias (Deschler and Lubbert,2006). The 5-year overall survival (OS) in patients older than 75 isless than 10% with no improvement over the last 30 years. The 5-year OSfor patients aged 25 to 39 has improved from less than 10% to 50%(Showel and Levis, 2014). The mortality rate for males is higher thanfor females. AML mortality is greater in whites than in blacks (Deschlerand Lubbert, 2006).

AML is diagnosed when at least 20% (World Health Organization (WHO)classification) or at least 30% (French-American-British (FAB)classification) blasts of the myeloid lineage are present in bone marrowor blood. Diagnosis is done on blood or bone marrow samples. Testsinclude a complete blood count and microscopic exams, cytochemistry,immunohistochemistry, flow cytometry, reverse transcriptase polymerasechain reaction (RT-PCR) and fluorescence in-situ hybridization (FISH)(National Cancer Institute, 2015).

Symptoms for AML are often unspecific and include weight loss, loss ofappetite, fatigue, fever, headaches and sleepiness. Risk factors fordeveloping AML include smoking, male gender, exposure to benzene,chemotherapeutical treatment with alkylating agents or topoisomerase IIinhibitors and radiation exposure (National Cancer Institute, 2015).

As AML is a very heterogenous disease there is no unique classificationsystem. The WHO classifies AML according to morphology, cytogenetics,molecular genetics and immunologic markers. The FAB system classifiesAML using morphology as determined by the degree of differentiation andthe extend of cell maturation. Depending on the above-named criteria thefollowing AML subtypes exist according to WHO and FAB (National CancerInstitute, 2015):

WHO:

Percentage Classification Description of AML AML with AML witht(8;21)(q22;q22) (AML/ETO)  5-12% characteristic AML withinv(16)(p13q22) or t(16;16)(p13;q22) 10-12% genetic (CBF□/MYH11)abnormalities APL: AML with t(15;17)(q22;q12) (PML/RARA  5-8% andvariants) AML with 11q23 (MLL) abnormalities  5-6% AML with AML withFLT3 mutations (FLT3/ITD: 23%, 20-30% genetic FLT3 point mutation: 7%)abnormalities AML with NPM1 mutations in FLT3, AML with CEBPA mutationsNPM1, AML with t(9;11)(p22;q23) (MLLT3/MLL) CEBPA, AML witht(6;9)(p23;q34) (DEK/NUP214) MLLT3/MLL, AML with inv(3)(q21q26.2) ort(3;3)(q21;q26.2) DEK/NUP214, (RPN1/EVI1) RPN1/EVI1, AML witht(1;22)(p13;q13) (RBM15/MKL1) RBM15/MKL1 AML with AML evolving from MDSor following MDS, multilineage variants: AML with complex karyotype, AMLdysplasia with deletions/aberrations of/in the following chromosomes:del(7q), del(5q), i(17q)7t(17p), del(13q), del(11q), del(12p)/t(12p),del(9q), idic(X)(q13), AML with translocations between the followingchromosomes: t(11;16)(q23;q13.3), t(3;21)(q26.2;q22.1),t(1;3)(p36.3;q21.1), t(2;11)(p21;q23), t(5;12)(q33;p12),t(5;7)(q33;q11.2), t(5;17)(q33;p13), t(5;10)(q33;q21), t(3;5)(q25;q34)AML and Alkylating agent-related AML and MDS MDS, therapy Topoisomerase11 inhibitor-related AML related AML not Acute myeloblastic leukemia,minimally    5% otherwise differentiated categorized Acute myeloblasticleukemia, without maturation   10% Acute myeloblastic leukemia, withmaturation 30-45% Acute myelomonocytic leukemia (AMML) 15-25% Acutemonoblastic leukemia and acute  5-8% monocytic leukemia Acute erythroidleukemias  5-6% Acute megakaryoblastic leukemia, variant:  3-5%transient myeloproliferative disorder in Down syndrome Acute basophilicleukemia  <1% Acute panmyelosis with myelofibrosis Myeloid sarcoma  2-8%Acute Acute leukemia of undetermined lineage leukemias of Acute leukemiawith mixed phenotype ambiguous Acute leukemia with mixed lineage lineageHybrid acute leukemiaFAB:

Classification Name Cytogenetics M0 acute myeloblastic leukemia,minimally differentiated M1 acute myeloblastic leukemia, withoutmaturation M2 acute myeloblastic leukemia, with t(8;21)(q22;q22),maturation t(6;9) M3 acute promyelocytic leukemia (APL) t(15;17) M4acute myelomonocytic leukemia (AMML) inv(16)(p13q22), del(16q) M4(Eo)acute myelomonocytic leukemia with inv(16), t(16;16) bone marroweosinophilia M5a and acute monoblastic leukemia and acute del(11q),t(9;11), M5b monocytic leukemia t(11;19) M6a and acute erythroidleukemias M6b M7 acute megakaryoblastic leukemia t(1;22) M8 acutebasophilic leukemia

AML treatment is divided into two phases: induction therapy andpost-remission/“consolidation therapy”. Induction therapy isadministered to induce remission and consists of combinationalchemotherapy. Consolidation therapy consists of additional chemotherapyor hematopoietic cell transplantation (HCT) (Showel and Levis, 2014).

The most common chemotherapeutic drugs used to treat AML are cytarabine,daunorubicin, idarubicin and mitoxantrone followed by cladribine,fludarabine and diverse others. Azacitidine and decitabine (DNAhypomethylating agents) are now used for treatment of MDS/AML. Treatmentfor APL/AML M3 includes all-trans retinoic acid (ATRA) and arsenictrioxide (ATO) (National Cancer Institute, 2015).

“Standard treatment” for AML is considered as “3+7”: 3 days ofidarubicin or daunorubicin and 7 days of cytarabine, followed by severalsimilar courses to achieve complete remission (CR) (Estey, 2014). Thedecision between standard therapy and clinical trial is based on therisk stratification. The European Leukemia Net (ELN) systemdistinguishes between the following prognostic groups:

Prognistic goup Subsets “Favorable” inv(16) or t(16;16); t(8;21); NK andNPM1+/FLT3 ITD−; NK and CEBPA+/+ Intermediate-1 NK and NPM1−/FLT3 ITD−;CEBPA+/− Intermediate-2 Cytogenetic abnormalities not in “favorable” or“adverse” groups; FLT3 ITD+ “Adverse” −5, −7, 5q-, abn 3q, 17p, 11q(other than 9;11), t(6;9), complex; insufficient metaphases for analysis

AML cases with intermediate-risk karyotype show either no karyotypicabnormalities or only one or two abnormalities not categorized as high-or low-risk.

FLT3 mutations are associated with an aggressive type of AML and a poorprognosis. They often occur together with NPM1 and DNMT3a (DNAmethyltransferase 3A) mutations. NPM1 (nucleophosmin) mutations are afavorable prognostic indicator, if not found together with FLT3mutations. CEPBA (CCAAT-enhancer-binding protein alpha/C/EBPα) mutationsconfer a survival advantage in the case of double or homozygous CEBPAmutations without wild-type expression. Altered methylation patterns ina variety of genes are caused by mutations in isocitrate dehydrogenase(IDH1 and IDH2) and DNMT3A. These mutations are associated with poorsurvival.

AML cases with favorable-risk karyotype consist of APL (acutepromyelocytic leukemia) and CBF (core-binding factor) leukemias. APLcases are associated with the fusion of the myeloid transcription factorPML to the retinoic acid receptor subunit alpha (RARA). The PML/RARAtranslocation is a favorable prognostic mutation. CBF leukemia casesshow translocations involving a subunit of CBF. In t(8;21) CBF alpha isfused to the ETO gene. In inv(16) CBF beta is fused to the smooth musclemyosin heavy chain. CBF translocations are very favorable prognosticmutations.

AML cases with unfavorable-risk karyotype are characterized by a complexkaryotype including chromosomal aberrations such as translocations,unbalanced rearrangements and gains/losses of whole chromosomes. Theyare associated with a poor prognosis.

MDS/AML cases evolve from myelodysplastic syndromes and carry a worseprognosis than other AML sub-groups (Showel and Levis, 2014).

Besides the above-listed prognostic factors, additional molecularmarkers or marker combinations can be used to judge the prognosis inspecific cytogenetic subsets:

TP53 mutations are the most unfavorable genetic alteration in AML. NPM1mutated and FLT3 WT together with a mutation in IDH1 or IDH2 is seen asfavorable. Unfavorable factors include a partial tandem duplication inthe MLL gene, a mutated TET2 gene, FLT3 ITD+together with a mutation inDNMT3a and CEBPA, FLT3 ITD—together with a mutation in ASXL1 or PHF6,and CD25 expression (stem cell-like “signature” and poorer outcome). Thepresence of CKIT mutations converts the prognosis of patients with afavorable inv(16) or t(8;21) into a more intermediate range. SPARC isup-regulated in NK (normal karyotype) patients with unfavorable geneexpression signature and down-regulated in association with thefavorable NPM1 mutation. miR-155 over-expression conveys a poorprognosis in NK AML. Differentially methylated regions (DMRs) areprognostic when found in association with several genes (FLT3 mutation,NPM1 mutation). In this case, a lower expression is associated with abetter prognosis (Estey, 2014).

Post-treatment information/information about minimal residual disease(MRD) should be included into following treatment decisions. Theseinclude the response to induction therapy, PCR of fusion transcripts,mutated genes and over-expressed genes to detect MRD and multi-parameterflow cytometry for observation of aberrant expression of surfaceantigens.

The following table combines AML prognostic groups and treatmentrecommendations:

Prognostic group Subsets Induction Post-remission “Favorable” inv(16) ort(16;16); 3 + 7; consider ara-C (6 doses × 2 t(8;21); NK and FLAG-ida ifage courses), possibly NPM1+/FLT3 ITD−; <60-65 preceded by 1 NK andCEBPA+/+ course FLAG-ida Intermediate-1/ NK and NPM1−/ 3 + 7; consider(a) HCT from matched Int-1 FLT3 ITD−; FLAG-ida if age sibling donor(MSD) CEBPA+/− <60-65 or (b) clinical if risk NRM <20- trial 25%; if notHCT candidate FLAG-ida then ara-C or clinical trial Intermediate-2/Cytogenetic 3 + 7; consider (a) HCT from MSD or Int-2 abnormalities notin FLAG-ida if age URD if risk NRM “favorable” or <60-65 or (b) clinical<30%; otherwise as “adverse” groups; trial; clinical trialintermediate-1; if FLT3 ITD+ combining FLT3+/FLT3 ITD+ chemotherapy withconsider FLT3 FLT3 inhibtor (e.g. inhibitor post HCT quizartinib,crenolanib) “Adverse” −5, −7, 5q-, abn 3q, Clinical trial HCT from MSDor 17p, 11q (other than URD if risk NRM 9;11), t(6;9), <40%; considerpost- complex; HCT trial; if not insufficient HCT: candidate metaphasesfor clinical trial analysis NRM: non-relapse mortality after HCT

Treatment options for the prognostic groups favorable, intermediate-1and possibly intermediate-2 include the “standard therapy” “3+7”, acombination of daunorubicin and cytarabine or idarubicin and cytarabine,or the administration of FLAG-ida, a combination of fludarabine,cytarabine, G-CSF and idarubicin. A third option is gemtuzumabozogamicin (GO), which is a conjugate of an anti-CD33 monoclonalantibody and the cytotoxic agent calechiamicin. As CD33 is expressedin >90% of patients with AML, the use of GO in combination with 3+7 orFLAG-ida may lead to longer remissions and survival. Dasatinib is givenin combination with 3+7 and then in combination with high-dosecytarabine in patients with CKIT mutations in inv(16) and t(8;21) AML(Estey, 2014).

Clinical trials are recommended for patients who belong to theprognostic groups unfavorable and intermediate-2. Treatment optionsinclude hypomethylating agents (HMAs) as azacitidine or decitabine,CPX-351, which is a liposomal formulation of daunorubicin and cytarabinein a 1:5 “optimal” molar ratio, and volasertib, which is an inhibitor ofpolo kinases. Volasertib is given in combination with LDAC (low-dosecytarabine). Several different FLT3 inhibitors can be administered incase of FLT3 mutations. These include sorafenib, which is given incombination with 3+7, quizartinib, a more selective inhibitor of FLT3ITD that also inhibits CKIT, crenolanib, and midostaurin, an unselectiveFLT3 ITD inhibitor. Another treatment option is targeting CD33 withantibody-drug conjugates (anti-CD33+calechiamicin, SGN-CD33a,anti-CD33+actinium-225), bispecific antibodies (recognition of CD33+CD3(AMG 330) or CD33+CD16) and chimeric antigen receptors (CARs) (Estey,2014).

If possible, AML patients can undergo allogeneic hematopoietic celltransplantation (HCT). HCT in CR (complete remission) 1 should beperformed if the expected reduction in risk of relapse is greater thanthe risk of HCT-related complications. HCT can also be performed in CR2or with active disease, as some patients who relapse after not receivingHCT in CR1 can still be cured. Patients that are seen unfit formyeloablative (MA) intensity HCT (patients>70-75, frail youngerpatients) can undergo a reduced intensity HCT (RIC-HCT). Possible donorsare MUDs (matched unrelated donors), haploidentical donors or cells fromumbilical cords that exceed the number of MSD (matched sibling donors).One third of patients have a HLA-matched sibling and about 50% have apotential matched unrelated donor (MUD) (Estey, 2014).

Considering the severe side-effects and expense associated with treatingcancer, there is a need to identify factors that can be used in thetreatment of cancer in general and AML in particular. There is also aneed to identify factors representing biomarkers for cancer in generaland AML in particular, leading to better diagnosis of cancer, assessmentof prognosis, and prediction of treatment success.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens.

The current classification of tumor associated antigens (TAAs) comprisesthe following major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor-(-associated) exon in case of proteins with tumor-specific(-associated) isoforms.e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.This non-classical way of class I presentation is referred to ascross-presentation in the literature (Brossart and Bevan, 1997; Rock etal., 1990). MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs e.g. during endocytosis, and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positive Tcells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of great importance for the development ofpharmaceutical products for triggering anti-tumor immune responses(Gnjatic et al., 2003). At the tumor site, T helper cells, support acytotoxic T cell− (CTL−) friendly cytokine milieu (Mortara et al., 2006)and attract effector cells, e.g. CTLs, natural killer (NK) cells,macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006).

Elongated (longer) peptides of the invention can act as MHC class IIactive epitopes.

T-helper cells, activated by MHC class II epitopes, play an importantrole in orchestrating the effector function of CTLs in anti-tumorimmunity. T-helper cell epitopes that trigger a T-helper cell responseof the TH1 type support effector functions of CD8-positive killer Tcells, which include cytotoxic functions directed against tumor cellsdisplaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNγ) (Beatty andPaterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cellsas direct anti-tumor effectors (Braumuller et al., 2013; Tran et al.,2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T cells (ligand: MHC class I molecule+peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-1-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e. copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g. in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated and thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). It isessential that epitopes are present in the amino acid sequence of theantigen, in order to ensure that such a peptide (“immunogenic peptide”),being derived from a tumor associated antigen, leads to an in vitro orin vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a T cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T-cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy. This is because only an individual subpopulation of epitopes ofthese antigens are suitable for such an application since a T cell witha corresponding TCR has to be present and the immunological tolerancefor this particular epitope needs to be absent or minimal. In a verypreferred embodiment of the invention it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T cell can be found. Such afunctional T cell is defined as a T cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 188 or a variant sequencethereof which is at least 77%, preferably at least 88%, homologous(preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 toSEQ ID NO: 188, wherein said variant binds to MHC and/or induces T cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 188 or a variant thereof, whichis at least 77%, preferably at least 88%, homologous (preferably atleast 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 188,wherein said peptide or variant thereof has an overall length of between8 and 100, preferably between 8 and 30, and most preferred of between 8and 14 amino acids.

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, and the prospective source(underlying) genes for these peptides. All peptides in Table 1 and Table2 bind to HLA-A*02. The peptides in Table 2 have been disclosed beforein large listings as results of high-throughput screenings with higherror rates or calculated using algorithms, but have not been associatedwith cancer at all before. The peptides in Table 3 are additionalpeptides that may be useful in combination with the other peptides ofthe invention. The peptides in Table 4 are furthermore useful in thediagnosis and/or treatment of various other malignancies that involve anover-expression or over-presentation of the respective underlyingpolypeptide.

TABLE 1 Peptides according to the present invention. J = phospho-serineSEQ ID No. Sequence GeneID(s) Official Gene Symbol(s)   1 LLDSAVYYL 2769GNA15   2 VLLKAVAQA 3978 LIG1   3 ALYDKTKRIFL 1791 DNTT   4 FLPDAFVTM1791 DNTT   5 FLYYEDLVSC 1791 DNTT   6 GLAEVLLAA 4261 CIITA   7LLWGDIMEL 79009 DDX50   8 LLWPGAALLV 7462 LAT2   9 SLLAYLEQA 57617 VPS18 10 VILDPVHSV 653659, 92703 TMEM183B, TMEM183A  11 ILTQIDHIL 26574 AATF 12 ALIESNTAL 255631 COL24A1  13 ALVPGVTQV 64745 METTL17  14 ALWWGTITL56479 KCNQ5  15 FIDEEVEDMYL 51244 CCDC174  16 FLDTQAPSL 116931 MED12L 17 FLLGLSEQL 389119 FAM212A  18 GIIEENWQL 221143 N6AMT2  19 GIVEYLSLV5557 PRIM1  20 GLDAFLLEL 57674 RNF213  21 GLFHGTELL 5291 PIK3CB  22GLLQLDTAFV 116115 ZNF526  23 GLLQPPVRIV 55749 CCAR1  24 GLVELLNRV 10636RGS14  25 GVEGSLIVEKI 3329 HSPD1  26 NAGVEGSLIVEKI 3329 HSPD1  27KANPALYVL 10594 PRPF8  28 LLDQMETPL 1511 CTSG  29 RLGPSVVGL 22984 PDCD11 30 SIISDSSAL 672 BRCA1  31 SLFVFIPMV 78992 YIPF2  32 SLSDRSWHL 29970SCHIP1  33 TIMNQEKLAKL 643412, 652963, BTF3P16, BTF3P12, BTF3 689  34TLSPWSFLI 79465 ULBP3  35 VLFEHAVGYAL 10528 NOP56  36 VLGPSPSSV 57597BAHCC1  37 VVAPAPVVEAV 3609 ILF3  38 AAIASTPTL 79612 NAA16  39 AIFAGTMQL55144 LRRC8D  40 ALAAGGYDVEKN 3009 HIST1H1B  41 ALFILPFVSV 10721 POLQ 42 ALTTYTIEV 54549 SDK2  43 AMLDFVSSL 55732 C1orf112  44 FAVDNVGNRTL55105 GPATCH2  45 FLFTDVLLM 84904 ARHGEF39  46 GLDQYLQEV 79825 CCDC48 47 GLIJPNVQL 79733 E2F8  48 IAIEALTQL 200424 TET3  49 IIDDNHAIV345645, 5700 PSMC1P4, PSMC1  50 IIWATSLLL 55728 N4BP2  51 SLLSSSLNV55728 N4BP2  52 IVDPVDSTL 5591 PRKDC  53 KAFLGELTL 257218 SHPRH  54KLPEFLVQL 203430 ZCCHC5  55 KTLDLINKL 57650 KIAA1524  56 LANPTTSAL 8295TRRAP  57 LLDFGSLSNLQV 100505503, RPS17L, RPS17P16, RPS17   402057, 6218 58 LLLATLQEA 790955 C11orf83  59 LSVPEGAIVSL 28672 TRAV12-3  60NLLNVLEYL 167127 UGT3A2  61 FLLPGVLLSEA 167127 UGT3A2  62 RLLFNLSEV253714 MMS22L  63 RLNDTIQLL 29128 UHRF1  64 SLANIKIWV 79895 ATP8B4  65SLEEQLSALTL 8458 TTF2  66 SLKNEVGGLV 121278 TPH2  67 SLQDRVIAL 145508CEP128  68 TGITTPVASV 23269 MGA  69 TIIGLVRVI 4998 ORC1  70 TLTDSNAQL64105 CENPK  71 TLTSSLATV 79571 GCC1  72 VAFPSGDASSL 23312 DMXL2  73VAIPDVDPL 777 CACNA1E  74 VANPVLYVL 11251 PTGDR2  75 VLAPLGFTL116150, 729148 NUS1, NUS1P1  76 VLLJPVPEL 64682 ANAPC1  77 VLNMKPPEI10592 SMC2  78 VLSEVECHL 261734 NPHP4  79 YLMDPDTFTF 9582 APOBEC3B  80YLTEALQSI 5427 POLE2  81 YVTEELPQL 2098 ESD  82 LLPDNFIAA 2098 ESD  83GLGAGVAEAV 6576 SLC25A1  84 GLLGSVLTI 8204 NRIP1  85 GLVPFGLYL 65250C5orf42  86 HLLGDPMANV 10897 YIF1A  87 ILKPFGNSI 3930 LBR  88 LALNFGSTL201266 SLC39A11  89 LLESPVDGWQV 54892 NCAPG2  90 LLLDTVISI 4130 MAP1A 91 RLAHYIDRV 84823 LMNB2  92 RLWDIQHQL 151790 WDR49  93 SLINDVLAE340554 ZC3H12B  94 SLLEFAQYL 2175 FANCA  95 SVAEINVLI 221527 ZBTB12  96TLLASYVFL 201305 SPNS3  97 TIMTGVIGV 201305 SPNS3  98 TQFGFLMEV 84674CARD6  99 YLAPFSLSNY 79968 WDR76 100 AAPAVLGEVDTSLV 4353 MPO 101AINKDPEAPIFQV 2108 ETFA 102 ALAQGAERV 23630 KCNE1L 103 ALGDFGIRL 114548NLRP3 104 ALIPETTTL 100529251, CKLF-CMTM1, CKLF 51192 105 GVFALVTAV100529251, CKLF-CMTM1, CKLF 51192 106 ALLEELERSTL 9404 LPXN 107ALLGMLPLL 8698 S1PR4 108 ELEMNSDLKAQL 100128060, 6201 RPS7P10, RPS7 109GLLAVPLLAA 4232 MEST 110 GLTHTAVVPLDLV 5250 SLC25A3 111 GVEPAADGKGVVVV6158 RPL28 112 ILRDALNQA 6238 RRBP1 113 NLQSEVEGV 8673 VAMP8 114RLAQEAAQV 114822 RHPN1 115 SLPDLTTPL 55904 MLL5 116 TILEILPEL 1462 VCAN117 TILPTILFL 54497 HEATR5B 118 TLLTVLTQA 57508 INTS2 119 TLTDELAAL51170 HSD17B11 120 VIQDLVVSV 23556 PIGN 121 VLQAGQYGV 1786 DNMT1 122VLYLEEVLL 9392 TGFBRAP1 123 YTVKINPTL 219972 MPEG1 124 GLPELVIQL 23279NUP160 125 GLFGYLVFL 10312 TCIRG1 126 GLLPQQIQAV 25777 SUN2 127KIISALPQL 152579 SCFD2 128 NLSTKTEAV 140890 SREK1 129 RMAVLNEQV 4678NASP 130 GVLGNALEGV 4678 NASP 131 SLFSGSLEPV 6733 SRPK2 132 SLYPVLNFL94005 PIGS 133 TVIGTLLFL 84876 ORAI1 134 AAGAGLPESV 9761 MLEC 135GIIDRIFQA 6894 TARBP1 136 GLSSIETLL 1602 DACH1 137 ILAPLAWDL 11270 NRM138 ILSDNLRQV 5987 TRIM27 139 NLIIFSPSV 1650 DDOST 140 YIPDFLTLL 64860ARMCX5 141 GLLPPLRIPELL 79171 RBM42 142 GLSDGYGFTT 3185 HNRNPF 143YLLPHILVY 545 ATR 144 GLFMGLVLV 56255 TMX4 145 VLLPLIFTI 146722 CD300LF146 ALDTRVVEL 6320 CLEC11A 147 FALPILNAL 51202 DDX47 148 FLYFEDHGL 55255WDR41 149 GLAEILVLV 9903 KLHL21 150 GLFGVLNEI 8086 AAAS 151 GLLPFPEVTL25909, 285116 AHCTF1, AHCTF1P1 152 GLSNHIAAL 29028 ATAD2 153 GLYTGQLAL706 TSPO 154 IIADNIIFL 6742 SSBP1 155 ILDLIQVFV 100526783, C15orf38-91023, 1176, AP3S2, AP3S2, AP3S1, 348110 C15orf38 156 ILMPELASL 5330PLCB2 157 ILTETQQGL 26057 ANKRD17 158 LLLGGTALA 1991 ELANE 159 LLPLAPAAA84519 ACRBP 160 RLVPFLVFV 3954 LETM1 161 SLIGIAIAL 9056 SLC7A7 162SLLDFLTFA 23317 DNAJC13 163 SLMIDLIEV 57488 ESYT2 164 SLNPQEDVEF 84365MK167IP 165 SLVDRVAAA 4883 NPR3 166 VLFPLNLQL 1378, 1379 CR1, CR1L 167VLLDVALGL 9091 PIGQ 168 VLLFETALL 3326, 3327 HSP90AB1, HSP90AB3P 169VLQDPIWLL 5187, 8864 PER1, PER2

TABLE 2 Additional peptides according to the present invention with noprior known cancer association. J = phospho-serine SEQ ID No. SequenceGeneID(s) Official Gene Symbol(s) 170 IVTEVAVGV  58155 PTBP2 171KLLKQVDFL  55272 IMP3 172 KLLWGDIMEL  79009 DDX50 173 KMQETLVGL   8379MAD1L1 174 NLTENLQYV  55344 PLCXD1 175 KMDJFLDMQL  55728 N4BP2 176HLWTGEEQL 146850 PIK3R6 177 KITTVIQHV  64946 CENPH 178 KLWPLFVKL  55183RIF1 179 RLISTLENL    641 BLM 180 ALDQEIIEV 100505503, RPS17L, RPS17P16,402057, 442216, RPS17P5, RPS17 6218 181 KLLNHVTQL  79075 DSCC1 182SLSEYDQCL   2769 GNA15 183 SVIGVSPAV   8567 MADD 184 RMTDQEAIQDL   4869NPM1 185 RLIPIIVLL  64005 MYO1G 186 IILDEAHNV   8458 TTF2 187 MLPPPPLTA  3978 LIG1 188 RLLDFPTLL  54627 KIAA1383

TABLE 3 Peptides useful for e.g. personalized cancer therapies. SEQOfficial ID No. Sequence GeneID(s) Gene Symbol(s) 189 RLFEEVLGV   9816URB2 190 SLYKGLLSV  25788 RAD54B 191 ALSVLRLAL   6691 SPINK2 192GLAALAVHL   2175 FANCA 193 FLLAEDTKV  10592 SMC2 194 LLWGNLPEI653820, 729533 FAM72B, FAM72A 195 FLFVDPELV 146850 PIK3R6 196 ILVDWLVQV  9133 CCNB2 197 VLLNEILEQV  64151 NCAPG 198 KIQEILTQV  10643 IGF2BP3

The present invention furthermore generally relates to the peptidesaccording to the present invention for use in the treatment ofproliferative diseases, such as, for example, bile duct cancer, braincancer, breast cancer, chronic lymphocytic leukemia, colon or rectumcancer, esophageal cancer, gallbladder cancer, liver cancer, melanoma,non-hodgkin lymphoma, non-small cell lung cancer, ovarian cancer,pancreatic cancer, prostate cancer, kidney cancer, small cell lungcancer, urinary bladder cancer, uterine cancer.

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 188. More preferred are thepeptides—alone or in combination—selected from the group consisting ofSEQ ID NO: 1 to SEQ ID NO: 100 (see Table 1), and their uses in theimmunotherapy of AML, bile duct cancer, brain cancer, breast cancer,chronic lymphocytic leukemia, colon or rectum cancer, esophageal cancer,gallbladder cancer, liver cancer, melanoma, non-hodgkin lymphoma,non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostatecancer, kidney cancer, small cell lung cancer, urinary bladder cancer,uterine cancer, and preferably AML.

As shown in the following Table 4, many of the peptides according to thepresent invention are also found on other tumor types and can, thus,also be used in the immunotherapy of other indications. Also refer toFIGS. 1A-1D and Example 1.

TABLE 4Peptides according to the present invention and their specific uses in otherproliferative diseases, especially in other cancerous diseases. The table shows forselected peptides on which additional tumor types they were found and either over-presented on more than 5% of the measured tumor samples, or presented on more than5% of the measured tumor samples with a ratio of geometric means tumor vs normaltissues being larger than 3. Over-presentation is defined as higher presentation onthe tumor sample as compared to the normal sample with highest presentation. Normaltissues against which over-presentation was tested were: adipose tissue, adrenalgland, artery, blood cells, bone marrow, brain, central nerve, colon, duodenum,esophagus, eye, gallbladder, heart, kidney, liver, lung, lymph node, pancreas,parathyroid gland, peripheral nerve, peritoneum, pituitary gland, pleura, rectum,salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, thyroidgland, trachea, ureter, urinary bladder, vein. SEQ ID No. SequenceOther relevant organs/diseases   1 LLDSAVYYLMelanoma, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer,Gallbladder Cancer, Bile Duct Cancer   6 GLAEVLLAA NHL, Uterine Cancer  7 LLWGDIMEL NHL   8 LLWPGAALLV NHL   9 SLLAYLEQA BRCA  10 VILDPVHSVSCLC, NHL, BRCA, Uterine Cancer  13 ALVPGVTQVCLL, NHL, Melanoma, Uterine Cancer  14 ALWWGTITL CLL, NHL  15FIDEEVEDMYL NHL, Urinary bladder cancer, GallbladderCancer, Bile Duct Cancer  20 GLDAFLLELNHL, Gallbladder Cancer, Bile Duct Cancer  23 GLLQPPVRIV HCC  24GLVELLNRV NHL  27 KANPALYVL CLL  28 LLDQMETPL Melanoma  29 RLGPSVVGLMelanoma  31 SLFVFIPMV Urinary bladder cancer  32 SLSDRSWHL Brain Cancer 33 TIMNQEKLAKL CRC, NHL, Uterine Cancer  34 TLSPWSFLINSCLC, Uterine Cancer  35 VLFEHAVGYAL CRC, CLL, NHL, BRCA, Melanoma,Urinary bladder cancer  36 VLGPSPSSV BRCA, Melanoma  37 VVAPAPVVEAV PC 43 AMLDFVSSL NHL  45 FLFTDVLLM NHL  49 IIDDNHAIV Melanoma  52 IVDPVDSTLMelanoma  56 LANPTTSAL CLL, Gallbladder Cancer, Bile Duct Cancer  75VLAPLGFTL BRCA  78 VLSEVECHL Melanoma  79 YLMDPDTFTF SCLC  80 YLTEALQSICLL, NHL Brain Cancer, HCC, PC, NHL, BRCA,  82 LLPDNFIAAMelanoma, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer,Gallbladder Cancer, Bile Duct Cancer  83 GLGAGVAEAV CLL  84 GLLGSVLTIHCC, NHL, BRCA, Urinary bladder cancer, Uterine Cancer  86 HLLGDPMANVSCLC, OC  90 LLLDTVTSI Brain Cancer, BRCA  93 SLINDVLAE CLL  94SLLEFAQYL NHL 103 ALGDFGIRL Melanoma, Esophageal Cancer,Gallbladder Cancer, Bile Duct Cancer 104 ALIPETTTL NHL, Melanoma 105GVFALVTAV NHL, BRCA, Melanoma, Uterine Cancer 106 ALLEELERSTL NHL 107ALLGMLPLL CLL, NHL 108 ELEMNSDLKAQL Uterine Cancer 112 ILRDALNQARCC, CLL, NHL, Melanoma 113 NLQSEVEGV PC, CLL 114 RLAQEAAQVRCC, OC, Uterine Cancer 115 SLPDLTTPL CLL, NHL, BRCA, Melanoma 116TILEILPEL RCC, Melanoma, Gallbladder Cancer, Bile Duct Cancer 117TILPTILFL CLL 118 TLLTVLTQA CLL, NHL 119 TLTDELAALGallbladder Cancer, Bile Duct Cancer 120 VIQDLVVSVMelanoma, Uterine Cancer 121 VLQAGQYGV CLL, NHL, Uterine Cancer 122VLYLEEVLL CLL, NHL, Uterine Cancer 123 YTVKINPTLGallbladder Cancer, Bile Duct Cancer 124 GLPELVIQLHCC, CLL, NHL, BRCA, Melanoma, Urinary bladder cancer, Uterine Cancer,Gallbladder Cancer, Bile Duct Cancer 125 GLFGYLVFL SCLC, PC 127KIISALPQL CLL, NHL, Esophageal Cancer, OC,Gallbladder Cancer, Bile Duct Cancer 128 NLSTKTEAVNHL, OC, Uterine Cancer 129 RMAVLNEQV SCLC, NHL 132 SLYPVLNFLUrinary bladder cancer, Uterine Cancer 133 TVIGTLLFL NHL 135 GIIDRIFQACRC, HCC, BRCA 136 GLSSIETLL BRCA 137 ILAPLAWDL CLL, NHL, Uterine Cancer138 ILSDNLRQV NSCLC, HCC, CLL, NHL, Melanoma,Esophageal Cancer, Urinary bladder cancer 140 YIPDFLTLL CLL, Melanoma141 GLLPPLRIPELL PC, CLL, Urinary bladder cancer, UterineCancer, Gallbladder Cancer, Bile Duct Cancer 142 GLSDGYGFTTCRC, PC, CLL, NHL, BRCA, Esophageal Cancer, OC, Urinary bladder cancer143 YLLPHILVY RCC, Melanoma 144 GLFMGLVLV NHL, BRCA, Melanoma 148FLYFEDHGL SCLC, CLL, NHL, Melanoma, Uterine Cancer 149 GLAEILVLVRCC, Brain Cancer, NHL, Melanoma 150 GLFGVLNEI CLL, NHL, Uterine Cancer151 GLLPFPEVTL CLL, NHL 152 GLSNHIAAL PC, NHL 154 IIADNIIFLSCLC, RCC, CRC, HCC, CLL, NHL, BRCA, Melanoma, Esophageal Cancer,OC, Urinary bladder cancer, UterineCancer, Gallbladder Cancer, Bile Duct Cancer 155 ILDLIQVFVRCC, CLL, NHL, Melanoma, Uterine Cancer 157 ILTETQQGLNSCLC, CRC, NHL, BRCA, Melanoma, Urinary bladder cancer 160 RLVPFLVFVCLL 161 SLIGIAIAL BRCA, Esophageal Cancer, GallbladderCancer, Bile Duct Cancer 162 SLLDFLTFA NHL 163 SLMIDLIEV Melanoma, OC166 VLFPLNLQL CLL 167 VLLDVALGL RCC, NHL, BRCA, Melanoma 168 VLLFETALLCLL, BRCA, Urinary bladder cancer 169 VLQDPIWLL CLL, NHL 170 IVTEVAVGVRCC, CLL, NHL, Melanoma, Uterine Cancer 172 KLLWGDIMELGallbladder Cancer, Bile Duct Cancer 173 KMQETLVGLBrain Cancer, PrC, NHL, BRCA, Melanoma, Esophageal Cancer,Gallbladder Cancer, Bile Duct Cancer 174 NLTENLQYV NHL 183 SVIGVSPAVBrain Cancer, NHL, BRCA, Melanoma 184 RMTDQEAIQDL NHL, Melanoma 185RLIPIIVLL SCLC, NHL, Gallbladder Cancer, Bile Duct Cancer 186 IILDEAHNVCLL, NHL, Melanoma, Uterine Cancer 187 MLPPPPLTASCLC, RCC, Brain Cancer, CLL, NHL, BRCA, Melanoma, Esophageal Cancer,Urinary bladder cancer, Uterine Cancer,Gallbladder Cancer, Bile Duct Cancer 188 RLLDFPTLLSCLC, RCC, CRC, HCC, CLL, NHL, BRCA, OC NSCLC = non-small cell lungcancer, SCLC = small cell lung cancer, RCC = kidney cancer, CRC = colonor rectum cancer, HCC = liver cancer, PC = pancreatic cancer, PrC =prostate cancer, BRCA = breast cancer, OC = ovarian cancer, CLL =chronic lymphocytic leukemia, NHL = non-hodgkin lymphoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 37, 82, 113, 125, 141, 142, and 152 for the—in onepreferred embodiment combined—treatment of pancreatic cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 32, 82, 90, 149, 173, 183, and 187 for the—in onepreferred embodiment combined—treatment of brain cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 15, 20, 56, 82, 103, 116, 119, 123, 124, 127, 141,154, 161, 172, 173, 185, and 187 for the—in one preferred embodimentcombined—treatment of gallbladder and bile duct cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 9, 10, 35, 36, 75, 82, 84, 90, 105, 115, 124, 135,136, 142, 144, 154, 157, 161, 167, 168, 173, 183, 187, and 188 forthe—in one preferred embodiment combined—treatment of breast cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 13, 14, 27, 35, 56, 80, 83, 93, 107, 112, 113, 115,117, 118, 121, 122, 124, 127, 137, 138, 140, 141, 142, 148, 150, 151,154, 155, 160, 166, 168, 169, 170, 186, 187, and 188 for the—in onepreferred embodiment combined—treatment of chronic lymphocytic leukemia.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 33, 35, 135, 142, 154, 157, and 188 for the—in onepreferred embodiment combined—treatment of colorectal cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 82, 103, 127, 138, 142, 154, 161, 173, and 187 forthe—in one preferred embodiment combined—treatment of esophageal cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 23, 82, 84, 124, 135, 138, 154, and 188 for the—in onepreferred embodiment combined—treatment of liver cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 13, 28, 29, 35, 36, 49, 52, 78, 82, 103, 104, 105,112, 115, 116, 120, 124, 138, 140, 143, 144, 148, 149, 154, 155, 157,163, 167, 170, 173, 183, 184, 186, and 187 for the—in one preferredembodiment combined—treatment of melanoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 6, 7, 8, 10, 13, 14, 15, 20, 24, 33, 35, 43, 45, 80,82, 84, 94, 104, 105, 106, 107, 112, 115, 118, 121, 122, 124, 127, 128,129, 133, 137, 138, 142, 144, 148, 149, 150, 151, 152, 154, 155, 157,162, 167, 169, 170, 173, 174, 183, 184, 185, 186, 187, and 188 forthe—in one preferred embodiment combined—treatment of non-hodgkinlymphoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 34, 138, and 157 for the—in one preferred embodimentcombined—treatment of non-small cell lung cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 86, 114, 127, 128, 142, 154, 163, and 188 for the—inone preferred embodiment combined—treatment of ovarian cancer.

Thus, another aspect of the present invention relates to the use of thepeptide according to the present invention according to SEQ ID No. 173for the treatment of prostate cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 112, 114, 116, 143, 149, 154, 155, 167, 170, 187, and188 for the—in one preferred embodiment combined—treatment of kidneycancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 10, 79, 86, 125, 129, 148, 154, 185, 187, and 188 forthe—in one preferred embodiment combined—treatment of small cell lungcancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 15, 31, 35, 82, 84, 124, 132, 138, 141, 142, 154,157, 168, and 187 for the—in one preferred embodiment combined—treatmentof urinary bladder cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 6, 10, 13, 33, 34, 82, 84, 105, 108, 114, 120, 121,122, 124, 128, 132, 137, 141, 148, 150, 154, 155, 170, 186, and 187 forthe—in one preferred embodiment combined—treatment of uterine cancer.

Thus, another aspect of the present invention relates to the use of thepeptides according to the present invention for the—preferablycombined—treatment of a proliferative disease selected from the group ofAML, bile duct cancer, brain cancer, breast cancer, chronic lymphocyticleukemia, colon or rectum cancer, esophageal cancer, gallbladder cancer,liver cancer, melanoma, non-hodgkin lymphoma, non-small cell lungcancer, ovarian cancer, pancreatic cancer, prostate cancer, kidneycancer, small cell lung cancer, urinary bladder cancer, uterine cancer.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or—in an elongatedform, such as a length-variant—MHC class-II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 188.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID No. 1 to SEQ ID No. 188, preferably containing SEQ IDNo. 1 to SEQ ID No. 100, or a variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the T cellreceptor or the antibody or other peptide- and/or peptide-MHC-bindingmolecules according to the present invention as a medicament or in themanufacture of a medicament. Preferably, said medicament is activeagainst cancer.

Preferably, said medicament is a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are AML, bile duct cancer, braincancer, breast cancer, chronic lymphocytic leukemia, colon or rectumcancer, esophageal cancer, gallbladder cancer, liver cancer, melanoma,non-hodgkin lymphoma, non-small cell lung cancer, ovarian cancer,pancreatic cancer, prostate cancer, kidney cancer, small cell lungcancer, urinary bladder cancer, uterine cancer, and preferably AMLcells.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”that can be used in the diagnosis of cancer, preferably AML. The markercan be over-presentation of the peptide(s) themselves, orover-expression of the corresponding gene(s). The markers may also beused to predict the probability of success of a treatment, preferably animmunotherapy, and most preferred an immunotherapy targeting the sametarget that is identified by the biomarker. For example, an antibody orsoluble TCR can be used to stain sections of the tumor to detect thepresence of a peptide of interest in complex with MHC.

Optionally the antibody carries a further effector function such as animmune stimulating domain or toxin.

The present invention also relates to the use of these novel targets inthe context of cancer treatment.

DETAILED DESCRIPTION OF THE INVENTION

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

As used herein and except as noted otherwise all terms are defined asgiven below.

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, 12, 13 or 14 or longer,and in case of MHC class II peptides (elongated variants of the peptidesof the invention) they can be as long as 15, 16, 17, 18, 19 or 20 ormore amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 5 Expression frequencies F of HLA-A*02 and HLA-A*24 and the mostfrequent HLA-DR serotypes. Frequencies are deduced from haplotypefrequencies Gf within the American population adapted from Mori et al.(Mori et al., 1997) employing the Hardy- Weinberg formula F = 1 −(1-Gf)². Combinations of A*02 or A*24 with certain HLA-DR alleles mightbe enriched or less frequent than expected from their single frequenciesdue to linkage disequilibrium. For details refer to Chanock et al.(Chanock et al., 2004) Calculated phenotype from Allele Populationallele frequency A*02 Caucasian (North America)  49.1% A*02 AfricanAmerican (North America)  34.1% A*02 Asian American (North America) 43.2% A*02 Latin American (North American)  48.3% DR1 Caucasian (NorthAmerica)  19.4% DR2 Caucasian (North America)  28.2% DR3 Caucasian(North America)  20.6% DR4 Caucasian (North America)  30.7% DR5Caucasian (North America)  23.3% DR6 Caucasian (North America)  26.7%DR7 Caucasian (North America)  24.8% DR8 Caucasian (North America)  5.7%DR9 Caucasian (North America)  2.1% DR1 African (North) American 13.20%DR2 African (North) American 29.80% DR3 African (North) American 24.80%DR4 African (North) American 11.10% DR5 African (North) American 31.10%DR6 African (North) American 33.70% DR7 African (North) American 19.20%DR8 African (North) American 12.10% DR9 African (North) American  5.80%DR1 Asian (North) American  6.80% DR2 Asian (North) American 33.80% DR3Asian (North) American  9.20% DR4 Asian (North) American 28.60% DR5Asian (North) American 30.00% DR6 Asian (North) American 25.10% DR7Asian (North) American 13.40% DR8 Asian (North) American 12.70% DR9Asian (North) American 18.60% DR1 Latin (North) American 15.30% DR2Latin (North) American 21.20% DR3 Latin (North) American 15.20% DR4Latin (North) American 36.80% DR5 Latin (North) American 20.00% DR6Latin (North) American 31.10% DR7 Latin (North) American 20.20% DR8Latin (North) American 18.60% DR9 Latin (North) American  2.10% A*24Philippines   65% A*24 Russia Nenets   61% A*24:02 Japan   59% A*24Malaysia   58% A*24:02 Philippines   54% A*24 India   47% A*24 SouthKorea   40% A*24 Sri Lanka   37% A*24 China   32% A*24:02 India   29%A*24 Australia West   22% A*24 USA   22% A*24 Russia Samara   20% A*24South America   20% A*24 Europe   18%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein bind to A*02. A vaccine may alsoinclude pan-binding MHC class II peptides. Therefore, the vaccine of theinvention can be used to treat cancer in patients that are A*02positive, whereas no selection for MHC class II allotypes is necessarydue to the pan-binding nature of these peptides.

If A*02 peptides of the invention are combined with peptides binding toanother allele, for example A*24, a higher percentage of any patientpopulation can be treated compared with addressing either MHC class Iallele alone. While in most populations less than 50% of patients couldbe addressed by either allele alone, a vaccine comprising HLA-A*24 andHLA-A*02 epitopes can treat at least 60% of patients in any relevantpopulation. Specifically, the following percentages of patients will bepositive for at least one of these alleles in various regions: USA 61%,Western Europe 62%, China 75%, South Korea 77%, Japan 86%.

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula:percent identity=100[1−(C/R)]wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and(ii) each gap in the Reference Sequence and(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and(iiii) the alignment has to start at position 1 of the alignedsequences;and R is the number of bases or amino acids in the Reference Sequenceover the length of the alignment with the Compared Sequence with any gapcreated in the Reference Sequence also being counted as a base or aminoacid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 188 or a variant thereof which is 88%homologous to SEQ ID NO: 1 to SEQ ID NO: 188, or a variant thereof thatwill induce T cells cross-reacting with said peptide. The peptides ofthe invention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 188. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T cells.

These T cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature and databases (Rammenseeet al., 1999; Godkin et al., 1997), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spatial properties of the polypeptidechains constituting the binding groove. Thus, one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO 188, by maintaining the known anchor residues, and wouldbe able to determine whether such variants maintain the ability to bindMHC class I or II molecules. The variants of the present inventionretain the ability to bind to the TCR of activated T cells, which cansubsequently cross-react with and kill cells that express a polypeptidecontaining the natural amino acid sequence of the cognate peptide asdefined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) class-I or-II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or —II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith other amino acid whose incorporation does not substantially affectT-cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 6 Variants and motif of the peptides according to SEQ ID NO: 1, 9,and 82 Position 1 2 3 4 5 6 7 8 9 SEQ ID NO. 1 L L D S A V Y Y LVariants A I V M M A M I M V A A A A I A V V V A V I V V T T A T I T V QQ A Q I Q V SEQ ID NO. 9 S L L A Y L E Q A Variants V I L M M V M I M LA A V A I A L V V V V I V L T T V T I T L Q Q V Q I Q L SEQ ID NO. 82 LL P D N F I A A Variants V I L M M V M I M L A A V A I A L V V V V I V LT T V T I T L Q Q V Q I Q L

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 11 amino acidslong, are generated by peptide processing from longer peptides orproteins that include the actual epitope. It is preferred that theresidues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in Table 7.

TABLE 7 Combinations of the elongations of peptides of the inventionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. This may lead to MHC class II binding peptides. Bindingto MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof between 8 and 100, preferably between 8 and 30, and most preferredbetween 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in caseof the elongated class II binding peptides the length can also be 15,16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 pM, andmost preferably no more than about 10 pM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 188.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO 188 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “Ii”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) (Meziere et al., 1997),incorporated herein by reference. This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. Meziere et al. (Meziere et al., 1997) showthat for MHC binding and T helper cell responses, these pseudopeptidesare useful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which isincorporated herein by reference. Chemical modification of amino acidsincludes but is not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995)for more extensive methodology relating to chemical modification ofproteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich provide information onspecific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. For example, diethylpyrocarbonate is areagent for the modification of histidyl residues in proteins. Histidinecan also be modified using 4-hydroxy-2-nonenal. The reaction of lysineresidues and other α-amino groups is, for example, useful in binding ofpeptides to surfaces or the cross-linking of proteins/peptides. Lysineis the site of attachment of poly(ethylene)glycol and the major site ofmodification in the glycosylation of proteins. Methionine residues inproteins can be modified with e.g. iodoacetamide, bromoethylamine, andchloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention. Generally,peptides and variants (at least those containing peptide linkagesbetween amino acid residues) may be synthesized by the Fmoc-polyamidemode of solid-phase peptide synthesis as disclosed by Lukas et al.(Lukas et al., 1981) and by references as cited therein. TemporaryN-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl(Fmoc) group. Repetitive cleavage of this highly base-labile protectinggroup is done using 20% piperidine in N, N-dimethylformamide. Side-chainfunctionalities may be protected as their butyl ethers (in the case ofserine threonine and tyrosine), butyl esters (in the case of glutamicacid and aspartic acid), butyloxycarbonyl derivative (in the case oflysine and histidine), trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N,N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated couplingprocedure. All coupling and deprotection reactions are monitored usingninhydrin, trinitrobenzene sulphonic acid or isotin test procedures.Upon completion of synthesis, peptides are cleaved from the resinsupport with concomitant removal of side-chain protecting groups bytreatment with 95% trifluoroacetic acid containing a 50% scavenger mix.Scavengers commonly used include ethanedithiol, phenol, anisole andwater, the exact choice depending on the constituent amino acids of thepeptide being synthesized. Also a combination of solid phase andsolution phase methodologies for the synthesis of peptides is possible(see, for example, (Bruckdorfer et al., 2004), and the references ascited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

In order to select over-presented peptides, a presentation profile iscalculated showing the median sample presentation as well as replicatevariation. The profile juxtaposes samples of the tumor entity ofinterest to a baseline of normal tissue samples. Each of these profilescan then be consolidated into an over-presentation score by calculatingthe p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015)adjusting for multiple testing by False Discovery Rate (Benjamini andHochberg, 1995) (cf. Example 1, FIGS. 1A through 1D).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESI) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of naturaltumor-associated peptides (TUMAPs) recorded from AML samples (N=17A*02-positive samples) with the fragmentation patterns of correspondingsynthetic reference peptides of identical sequences. Since the peptideswere directly identified as ligands of HLA molecules of primary tumors,these results provide direct evidence for the natural processing andpresentation of the identified peptides on primary cancer tissueobtained from 16 AML patients.

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

Presentation levels including error estimates for each peptide andsample were established. Peptides exclusively presented on tumor tissueand peptides over-presented in tumor versus non-cancerous tissues andorgans have been identified.

HLA-peptide complexes from AML tissue samples were purified andHLA-associated peptides were isolated and analyzed by LC-MS (seeexamples). All TUMAPs contained in the present application wereidentified with this approach on primary AML samples confirming theirpresentation on primary AML.

TUMAPs identified on multiple AML and normal tissues were quantifiedusing ion-counting of label-free LC-MS data. The method assumes thatLC-MS signal areas of a peptide correlate with its abundance in thesample. All quantitative signals of a peptide in various LC-MSexperiments were normalized based on central tendency, averaged persample and merged into a bar plot, called presentation profile. Thepresentation profile consolidates different analysis methods likeprotein database search, spectral clustering, charge state deconvolution(decharging) and retention time alignment and normalization.

Besides over-presentation of the peptide, mRNA expression of theunderlying gene was tested. mRNA data were obtained via RNASeq analysesof normal tissues and cancer tissues (cf. Example 2, FIGS. 2A through2C). An additional source of normal tissue data was a database ofpublicly available RNA expression data from around 3000 normal tissuesamples (Lonsdale, 2013). Peptides which are derived from proteins whosecoding mRNA is highly expressed in cancer tissue, but very low or absentin vital normal tissues, were preferably included in the presentinvention.

The present invention provides peptides that are useful in treatingcancers/tumors, preferably AML that over- or exclusively present thepeptides of the invention. These peptides were shown by massspectrometry to be naturally presented by HLA molecules on primary humanAML samples.

Many of the source gene/proteins (also designated “full-length proteins”or “underlying proteins”) from which the peptides are derived were shownto be highly over-expressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy bone marrow and blood cells or other normal tissue cells,demonstrating a high degree of tumor association of the source genes(see Example 2). Moreover, the peptides themselves are stronglyover-presented on tumor tissue—“tumor tissue” in relation to thisinvention shall mean a sample from a patient suffering from AML, but noton normal tissues (see Example 1).

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. AML cells presenting the derived peptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention (see Example 3, Example 4).Furthermore, the peptides when complexed with the respective MHC can beused for the production of antibodies and/or TCRs, in particular sTCRs,according to the present invention, as well. Respective methods are wellknown to the person of skill, and can be found in the respectiveliterature as well. Thus, the peptides of the present invention areuseful for generating an immune response in a patient by which tumorcells can be destroyed. An immune response in a patient can be inducedby direct administration of the described peptides or suitable precursorsubstances (e.g. elongated peptides, proteins, or nucleic acids encodingthese peptides) to the patient, ideally in combination with an agentenhancing the immunogenicity (i.e. an adjuvant). The immune responseoriginating from such a therapeutic vaccination can be expected to behighly specific against tumor cells because the target peptides of thepresent invention are not presented on normal tissues in comparable copynumbers, preventing the risk of undesired autoimmune reactions againstnormal cells in the patient.

The present description further relates to T-cell receptors (TCRs)comprising an alpha chain and a beta chain (“alpha/beta TCRs”). Alsoprovided are peptides according to the invention capable of binding toTCRs and antibodies when presented by an MHC molecule. The presentdescription also relates to nucleic acids, vectors and host cells forexpressing TCRs and peptides of the present description; and methods ofusing the same.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.The term also includes so-called gamma/delta TCRs.

In one embodiment the description provides a method of producing a TCRas described herein, the method comprising culturing a host cell capableof expressing the TCR under conditions suitable to promote expression ofthe TCR.

The description in another aspect relates to methods according to thedescription, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell or the antigen is loadedonto class I or II MHC tetramers by tetramerizing the antigen/class I orII MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region (V), and joining region(J). The variable domain may also include a leader region (L). Beta anddelta chains may also include a diversity region (D). The alpha and betaconstant domains may also include C-terminal transmembrane (TM) domainsthat anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term “TCR gamma variable domain”as used herein refers to the concatenation of the TCR gamma V (TRGV)region without leader region (L), and the TCR gamma J (TRGJ) region, andthe term TCR gamma constant domain refers to the extracellular TRGCregion, or to a C-terminal truncated TRGC sequence. Likewise the term“TCR delta variable domain” refers to the concatenation of the TCR deltaV (TRDV) region without leader region (L) and the TCR delta D/J(TRDD/TRDJ) region, and the term “TCR delta constant domain” refers tothe extracellular TRDC region, or to a C-terminal truncated TRDCsequence.

TCRs of the present description preferably bind to an peptide-HLAmolecule complex with a binding affinity (KD) of about 100 μM or less,about 50 μM or less, about 25 μM or less, or about 10 μM or less. Morepreferred are high affinity TCRs having binding affinities of about 1 μMor less, about 100 nM or less, about 50 nM or less, about 25 nM or less.Non-limiting examples of preferred binding affinity ranges for TCRs ofthe present invention include about 1 nM to about 10 nM; about 10 nM toabout 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40 nM;about 40 nM to about 50 nM; about 50 nM to about 60 nM; about 60 nM toabout 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; andabout 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description,“specific binding” and grammatical variants thereof are used to mean aTCR having a binding affinity (KD) for a peptide-HLA molecule complex of100 μM or less.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above,alpha/beta heterodimeric TCRs of the present description may have a TRACconstant domain sequence and a TRBC1 or TRBC2 constant domain sequence,and the TRAC constant domain sequence and the TRBC1 or TRBC2 constantdomain sequence of the TCR may be linked by the native disulfide bondbetween Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCRalpha chain and/or TCR beta chain has a binding affinity for, and/or abinding half-life for, a peptide-HLA molecule complex, which is at leastdouble that of a TCR comprising the unmutated TCR alpha chain and/orunmutated TCR beta chain. Affinity-enhancement of tumor-specific TCRs,and its exploitation, relies on the existence of a window for optimalTCR affinities. The existence of such a window is based on observationsthat TCRs specific for HLA-A2-restricted pathogens have KD values thatare generally about 10-fold lower when compared to TCRs specific forHLA-A2-restricted tumor-associated self-antigens. It is now known,although tumor antigens have the potential to be immunogenic, becausetumors arise from the individual's own cells only mutated proteins orproteins with altered translational processing will be seen as foreignby the immune system. Antigens that are upregulated or overexpressed (socalled self-antigens) will not necessarily induce a functional immuneresponse against the tumor: T-cells expressing TCRs that are highlyreactive to these antigens will have been negatively selected within thethymus in a process known as central tolerance, meaning that onlyT-cells with low-affinity TCRs for self-antigens remain. Therefore,affinity of TCRs or variants of the present description to peptides canbe enhanced by methods well known in the art.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*02-negative healthy donors withA2/peptide monomers, incubating the PBMCs with tetramer-phycoerythrin(PE) and isolating the high avidity T-cells by fluo-rescence activatedcell sorting (FACS)-Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRαβ geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with a peptide, incubating PBMCs obtained from the transgenic micewith tetramer-phycoerythrin (PE), and isolating the high avidity T-cellsby fluorescence activated cell sorting (FACS)-Calibur analysis.

In one aspect, to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding TCR-alpha and/or TCR-beta chains ofthe present description are cloned into expression vectors, such asgamma retrovirus or lentivirus. The recombinant viruses are generatedand then tested for functionality, such as antigen specificity andfunctional avidity. An aliquot of the final product is then used totransduce the target T-cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient.

In another aspect, to obtain T-cells expressing TCRs of the presentdescription, TCR RNAs are synthesized by techniques known in the art,e.g., in vitro transcription sys-tems. The in vitro-synthesized TCR RNAsare then introduced into primary CD8+ T-cells obtained from healthydonors by electroporation to re-express tumor specific TCR-alpha and/orTCR-beta chains.

To increase the expression, nucleic acids encoding TCRs of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter,elongation factor (EF)-1a and the spleen focus-forming virus (SFFV)promoter. In a preferred embodiment, the promoter is heterologous to thenucleic acid being expressed.

In addition to strong promoters, TCR expression cassettes of the presentdescription may contain additional elements that can enhance transgeneexpression, including a central polypurine tract (cPPT), which promotesthe nuclear translocation of lentiviral constructs (Follenzi et al.,2000), and the woodchuck hepatitis virus posttranscriptional regulatoryelement (wPRE), which increases the level of transgene expression byincreasing RNA stability (Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may beencoded by nucleic acids located in separate vectors, or may be encodedby polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bi-cistronic constructs in a singlevector, which has been shown to be capable of over-coming this obstacle.The use of a viral intraribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced (Schmitt et al., 2009).

Nucleic acids encoding TCRs of the present description may be codonoptimized to increase expression from a host cell. Redundancy in thegenetic code allows some amino acids to be encoded by more than onecodon, but certain codons are less “op-timal” than others because of therelative availability of matching tRNAs as well as other factors(Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta genesequences such that each amino acid is encoded by the optimal codon formammalian gene expression, as well as eliminating mRNA instabilitymotifs or cryptic splice sites, has been shown to significantly enhanceTCR-alpha and TCR-beta gene expression (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chainsmay result in the acquisition of specificities that pose a significantrisk for autoimmunity. For example, the formation of mixed TCR dimersmay reduce the number of CD3 molecules available to form properly pairedTCR complexes, and therefore can significantly decrease the functionalavidity of the cells expressing the introduced TCR (Kuball et al.,2007).

To reduce mispairing, the C-terminus domain of the introduced TCR chainsof the present description may be modified in order to promoteinterchain affinity, while de-creasing the ability of the introducedchains to pair with the endogenous TCR. These strategies may includereplacing the human TCR-alpha and TCR-beta C-terminus domains with theirmurine counterparts (murinized C-terminus domain); generating a secondinterchain disulfide bond in the C-terminus domain by introducing asecond cysteine residue into both the TCR-alpha and TCR-beta chains ofthe introduced TCR (cysteine modification); swapping interactingresidues in the TCR-alpha and TCR-beta chain C-terminus domains(“knob-in-hole”); and fusing the variable domains of the TCR-alpha andTCR-beta chains directly to CD3ζ (CD3ζ fusion) (Schmitt et al., 2009).

In an embodiment, a host cell is engineered to express a TCR of thepresent description. In preferred embodiments, the host cell is a humanT-cell or T-cell progenitor. In some embodiments the T-cell or T-cellprogenitor is obtained from a cancer patient. In other embodiments theT-cell or T-cell progenitor is obtained from a healthy donor. Host cellsof the present description can be allogeneic or autologous with respectto a patient to be treated. In one embodiment, the host is a gamma/deltaT-cell transformed to express an alpha/beta TCR.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides or TCR proteinseither in the free form or in the form of a pharmaceutically acceptablesalt (see also above). As used herein, “a pharmaceutically acceptablesalt” refers to a derivative of the disclosed peptides wherein thepeptide is modified by making acid or base salts of the agent. Forexample, acid salts are prepared from the free base (typically whereinthe neutral form of the drug has a neutral —NH2 group) involvingreaction with a suitable acid. Suitable acids for preparing acid saltsinclude both organic acids, e.g., acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinicacid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoicacid, cinnamic acid, mandelic acid, methane sulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, aswell as inorganic acids, e.g., hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid phosphoric acid and the like. Conversely,preparation of basic salts of acid moieties which may be present on apeptide are prepared using a pharmaceutically acceptable base such assodium hydroxide, potassium hydroxide, ammonium hydroxide, calciumhydroxide, trimethylamine or the like.

Another embodiment of the present invention relates to a non-naturallyoccurring peptide wherein said peptide consists or consists essentiallyof an amino acid sequence according to SEQ ID No: 1 to SEQ ID No: 188and has been synthetically produced (e.g. synthesized) as apharmaceutically acceptable salt. Methods to synthetically producepeptides are well known in the art. The salts of the peptides accordingto the present invention differ substantially from the peptides in theirstate(s) in vivo, as the peptides as generated in vivo are no salts. Thenon-natural salt form of the peptide mediates the solubility of thepeptide, in particular in the context of pharmaceutical compositionscomprising the peptides, e.g. the peptide vaccines as disclosed herein.A sufficient and at least substantial solubility of the peptide(s) isrequired in order to efficiently provide the peptides to the subject tobe treated. Preferably, the salts are pharmaceutically acceptable saltsof the peptides. These salts according to the invention include alkalineand earth alkaline salts such as salts of the Hofmeister seriescomprising as anions PO₄ ³⁻, SO₄ ²⁻, CH₃COO⁻, Cl⁻, Br⁻, NO₃ ⁻, ClO₄ ⁻,I⁻, SCN⁻ and as cations NH₄ ⁺, Rb⁺, K⁺, Na⁺, Cs⁺, Li⁺, Zn²⁺, Mg²⁺, Ca²⁺,Mn²⁺, Cu²⁺ and Ba²⁺. Particularly salts are selected from (NH₄)₃PO₄,(NH₄)₂HPO₄, (NH₄)H₂PO₄, (NH₄)₂SO₄, NH₄CH₃COO, NH₄Cl, NH₄Br, NH₄NO₃,NH₄ClO₄, NH₄I, NH₄SCN, Rb₃PO₄, Rb₂HPO₄, RbH₂PO₄, Rb₂SO₄, Rb₄CH₃COO,Rb₄Cl, Rb₄Br, Rb₄NO₃, Rb₄ClO₄, Rb₄I, Rb₄SCN, K₃PO₄, K₂HPO₄, KH₂PO₄,K₂SO₄, KCH₃COO, KCl, KBr, KNOB, KClO₄, KI, KSCN, Na₃PO₄, Na₂HPO₄,NaH₂PO₄, Na₂SO₄, NaCH₃COO, NaCl, NaBr, NaNO₃, NaClO₄, NaI, NaSCN, ZnCl₂Cs₃PO₄, Cs₂HPO₄, CsH₂PO₄, Cs₂SO₄, CsCH₃COO, CsCl, CsBr, CsNO₃, CsClO₄,CsI, CsSCN, Li₃PO₄, Li₂HPO₄, LiH₂PO₄, Li₂SO₄, LiCH₃COO, LiCl, LiBr,LiNO₃, LiClO₄, LiI, LiSCN, Cu₂SO₄, Mg₃(PO₄)₂, Mg₂HPO₄, Mg(H₂PO₄)₂,Mg₂SO₄, Mg(CH₃COO)₂, MgCl₂, MgBr₂, Mg(NO₃)₂, Mg(ClO₄)₂, MgI₂, Mg(SCN)₂,MnCl₂, Ca₃(PO₄), Ca₂HPO₄, Ca(H₂PO₄)₂, CaSO₄, Ca(CH₃COO)₂, CaCl₂, CaBr₂,Ca(NO₃)₂, Ca(ClO₄)₂, CaI₂, Ca(SCN)₂, Ba₃(PO₄)₂, Ba₂HPO₄, Ba(H₂PO₄)₂,BaSO₄, Ba(CH₃COO)₂, BaCl₂, BaBr₂, Ba(NO₃)₂, Ba(ClO₄)₂, BaI₂, andBa(SCN)₂. Particularly preferred are NH acetate, MgCl₂, KH₂PO₄, Na₂SO₄,KCl, NaCl, and CaCl₂), such as, for example, the chloride or acetate(trifluoroacetate) salts.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides or TCR proteins as salts of acetic acid(acetates), trifluoro acetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutic such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically i.d., i.m.,s.c., i.p. and i.v., or applied ex vivo to cells derived from thepatient or a human cell line which are subsequently administered to thepatient, or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin-2. The peptide may be substantially pure,or combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 T cells is more efficient in the presence of help provided by CD4T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 Tcells the fusion partner or sections of a hybrid molecule suitablyprovide epitopes which stimulate CD4-positive T cells. CD4- andCD8-stimulating epitopes are well known in the art and include thoseidentified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 188, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Saiki et al., 1988). This method may be used for introducingthe DNA into a suitable vector, for example by engineering in suitablerestriction sites, or it may be used to modify the DNA in other usefulways as is known in the art. If viral vectors are used, pox- oradenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3×FLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL, or may be linked without anyadditional peptide(s) between them. These constructs can also be usedfor cancer therapy, and may induce immune responses both involving MHC Iand MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbás and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (Cohen et al.,1972) and (Green and Sambrook, 2012). Transformation of yeast cells isdescribed in Sherman et al. (Sherman et al., 1986). The method of Beggs(Beggs, 1978) is also useful. With regard to vertebrate cells, reagentsuseful in transfecting such cells, for example calcium phosphate andDEAE-dextran or liposome formulations, are available from StratageneCloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877,USA. Electroporation is also useful for transforming and/or transfectingcells and is well known in the art for transforming yeast cell,bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantiallypure, or contained in a suitable vector or delivery system. The nucleicacid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g. Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T cellsand helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligandsderived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, saponin-based adjuvant (e.g., ISCOMATRIX®), ISCOMs,JuvImmune®, LipoVac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51,water-in-oil and oil-in-water emulsions, OK-432, OM-174, OM-197-MP-EC,Denileukin diftitox (ONTAK®), OspA, PepTel® vector system, poly(lactidco-glycolid) [PLG]-based and dextran microparticles, talactoferrinSRL172, Virosomes and other Virus-like particles, YF-17D, VEGF trap,R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which is derivedfrom saponin, mycobacterial extracts and synthetic bacterial cell wallmimics, and other proprietary adjuvants such as Ribi's Detox, Quil, orSuperfos. Adjuvants such as Freund's or GM-CSF are preferred. Severalimmunological adjuvants (e.g., MF59) specific for dendritic cells andtheir preparation have been described previously (Allison and Krummel,1995). Also cytokines may be used. Several cytokines have been directlylinked to influencing dendritic cell migration to lymphoid tissues(e.g., TNF-), accelerating the maturation of dendritic cells intoefficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF, IL-1and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporated herein byreference in its entirety) and acting as immunoadjuvants (e.g., IL-12,IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich et al., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g., CpR, IDERA®), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g., rintatolimod (AmpliGen®),poly-(ICLC) (Hiltonol®), poly(IC-R), poly(I:C12U), non-CpG bacterial DNAor RNA as well as immunoactive small molecules and antibodies such ascyclophosphamide, sunitinib, Bevacizumab®, celebrex, NCX-4016,sildenafil, tadalafil, vardenafil, sorafenib, temozolomide,temsirolimus, XL-999, CP-547632, pazopanib, VEGF Trap, ZD2171, AZD2171,anti-CTLA4, other antibodies targeting key structures of the immunesystem (e.g., anti-CD40, anti-TGFbeta, anti-TNFalpha receptor) andSC58175, which may act therapeutically and/or as an adjuvant. Theamounts and concentrations of adjuvants and additives useful in thecontext of the present description can readily be determined by theskilled artisan without undue experimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, poly-(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonal®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomatous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets. The composition isdesigned in such a way that each tumor is expected to express several ofthe antigens and cover several independent pathways necessary for tumorgrowth and maintenance. Thus, the vaccine can easily be used“off-the-shelf” for a larger patient population. This means that apre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment a scaffold is able toactivate signaling through its target antigen, for example a T cellreceptor complex antigen. Scaffolds include but are not limited toantibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting other peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e. not derived from the human HLA-peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, and anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 188,according to the invention at hand with the MHC molecule, using thecell-SELEX (Systematic Evolution of Ligands by Exponential enrichment)technique.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen, the method comprising: immunizing agenetically engineered non-human mammal comprising cells expressing saidhuman major histocompatibility complex (MHC) class I or II with asoluble form of a MHC class I or II molecule being complexed with saidHLA-restricted antigen; isolating mRNA molecules from antibody producingcells of said non-human mammal; producing a phage display librarydisplaying protein molecules encoded by said mRNA molecules; andisolating at least one phage from said phage display library, said atleast one phage displaying said antibody specifically binding to saidhuman major histocompatibility complex (MHC) class I or II beingcomplexed with said HLA-restricted antigen.

It is a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present invention are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isalso regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 188, ora variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 188 or a variant thereof thatinduces T cells cross-reacting with said peptide, wherein said peptideis not the underlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:188 or a variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 188, wherein said peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 188.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of AML.

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom said host cell or its culture medium.

The present invention further relates to the method according to thepresent invention, where-in the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 188 or said variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellsselectively recognizes a cell which aberrantly expresses a polypeptidecomprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are AML cells or other solid orhematological tumor cells such as bile duct cancer, brain cancer, breastcancer, chronic lymphocytic leukemia, colon or rectum cancer, esophagealcancer, gallbladder cancer, liver cancer, melanoma, non-hodgkinlymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer,prostate cancer, kidney cancer, small cell lung cancer, urinary bladdercancer, uterine cancer.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of AML. The present invention also relates to the use of thesenovel targets for cancer treatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of a AML marker (poly)peptide,delivery of a toxin to a AML cell expressing a cancer marker gene at anincreased level, and/or inhibiting the activity of a AML markerpolypeptide) according to the invention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length AML marker polypeptides or fragments thereof maybe used to generate the antibodies of the invention. A polypeptide to beused for generating an antibody of the invention may be partially orfully purified from a natural source, or may be produced usingrecombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 188polypeptide, or a variant or fragment thereof, can be expressed inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells), after which the recombinant protein can bepurified and used to generate a monoclonal or polyclonal antibodypreparation that specifically bind the AML marker polypeptide used togenerate the antibody according to the invention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed cancers or frozen tissuesections. After their initial in vitro characterization, antibodiesintended for therapeutic or in vivo diagnostic use are tested accordingto known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating AML, the efficacyof the therapeutic antibody can be assessed in various ways well knownto the skilled practitioner. For instance, the size, number, and/ordistribution of cancer in a subject receiving treatment may be monitoredusing standard tumor imaging techniques. A therapeutically-administeredantibody that arrests tumor growth, results in tumor shrinkage, and/orprevents the development of new tumors, compared to the disease coursethat would occurs in the absence of antibody administration, is anefficacious antibody for treatment of cancer.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), and domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it could be expressed inT cells used for adoptive transfer. Further information can be found inWO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs isdescribed in WO 2012/056407A1. Further methods for the production aredisclosed in WO 2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 188, or a variant aminoacid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Plebanski et al., 1995) madeuse of autologous peripheral blood lymphocytes (PLBs) in the preparationof T cells. Furthermore, the production of autologous T cells by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus is possible. Also, B cells can be used in theproduction of autologous T cells. In addition, macrophages pulsed withpeptide or polypeptide, or infected with recombinant virus, may be usedin the preparation of autologous T cells. S. Walter et al. (Walter etal., 2003) describe the in vitro priming of T cells by using artificialantigen presenting cells (aAPCs), which is also a suitable way forgenerating T cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC:peptidecomplexes to the surface of polystyrene particles (microbeads) bybiotin:streptavidin biochemistry. This system permits the exact controlof the MHC density on aAPCs, which allows to selectively eliciting high-or low-avidity antigen-specific T cell responses with high efficiencyfrom blood samples. Apart from MHC:peptide complexes, aAPCs should carryother proteins with co-stimulatory activity like anti-CD28 antibodiescoupled to their surface. Furthermore such aAPC-based systems oftenrequire the addition of appropriate soluble factors, e. g. cytokines,like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, andvaccinia-infected target cells. In addition plant viruses may be used(see, for example, Porta et al. (Porta et al., 1994) which describes thedevelopment of cowpea mosaic virus as a high-yielding system for thepresentation of foreign peptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 188.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to levels of expression in normal tissues orthat the gene is silent in the tissue from which the tumor is derivedbut in the tumor it is expressed. By “over-expressed” the inventors meanthat the polypeptide is present at a level at least 1.2-fold of thatpresent in normal tissue; preferably at least 2-fold, and morepreferably at least 5-fold or 10-fold the level present in normaltissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art. Reviews can be found in: Gattioni et al. and Morgan et al.(Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T-cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

The present invention is further directed at a kit comprising:

(a) a container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;

(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and

(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from AML, themedicament of the invention is preferably used to treat AML.

The present invention further relates to a method for producing apersonalized pharmaceutical for an individual patient comprisingmanufacturing a pharmaceutical composition comprising at least onepeptide selected from a warehouse of pre-screened TUMAPs, wherein the atleast one peptide used in the pharmaceutical composition is selected forsuitability in the individual patient. In one embodiment, thepharmaceutical composition is a vaccine. The method could also beadapted to produce T cell clones for down-stream applications, such asTCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group or set ofpeptides that have been pre-screened for immunogenicity and/orover-presentation in a particular tumor type. The term “warehouse” isnot intended to imply that the particular peptides included in thevaccine have been pre-manufactured and stored in a physical facility,although that possibility is contemplated. It is expressly contemplatedthat the peptides may be manufactured de novo for each individualizedvaccine produced, or may be pre-manufactured and stored. The warehouse(e.g. in the form of a database) is composed of tumor-associatedpeptides which were highly overexpressed in the tumor tissue of AMLpatients with various HLA-A HLA-B and HLA-C alleles. It may contain MHCclass I and MHC class II peptides or elongated MHC class I peptides. Inaddition to the tumor associated peptides collected from several AMLtissues, the warehouse may contain HLA-A*02 and HLA-A*24 markerpeptides. These peptides allow comparison of the magnitude of T-cellimmunity induced by TUMAPS in a quantitative manner and hence allowimportant conclusion to be drawn on the capacity of the vaccine toelicit anti-tumor responses. Secondly, they function as importantpositive control peptides derived from a “non-self” antigen in the casethat any vaccine-induced T-cell responses to TUMAPs derived from “self”antigens in a patient are not observed. And thirdly, it may allowconclusions to be drawn, regarding the status of immunocompetence of thepatient.

TUMAPs for the warehouse are identified by using an integratedfunctional genomics approach combining gene expression analysis, massspectrometry, and T-cell immunology (XPresident®). The approach assuresthat only TUMAPs truly present on a high percentage of tumors but not oronly minimally expressed on normal tissue, are chosen for furtheranalysis. For initial peptide selection, AML samples from patients andblood from healthy donors were analyzed in a stepwise approach:

1. HLA ligands from the malignant material were identified by massspectrometry

2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes over-expressed in the malignant tissue (AML)compared with a range of normal organs and tissues

3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T cells from healthy donors as well as fromAML patients.

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed, the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multitarget approach. Intheory an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In an aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patients tumor and, wherepossible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; (b) comparingthe peptides identified in (a) with a warehouse (database) of peptidesas described above; and (c) selecting at least one peptide from thewarehouse (database) that correlates with a tumor-associated peptideidentified in the patient. For example, the TUMAPs presented by thetumor sample are identified by: (a1) comparing expression data from thetumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. Preferably, the sequences of MHCligands are identified by eluting bound peptides from MHC moleculesisolated from the tumor sample, and sequencing the eluted ligands.Preferably, the tumor sample and the normal tissue are obtained from thesame patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine. As one example, candidate TUMAPsmay be identified in the patient by (a1) comparing expression data fromthe tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. As another example, proteins may beidentified containing mutations that are unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andTUMAPs can be identified that specifically target the mutation. Forexample, the genome of the tumor and of corresponding normal tissue canbe sequenced by whole genome sequencing: For discovery of non-synonymousmutations in the protein-coding regions of genes, genomic DNA and RNAare extracted from tumor tissues and normal non-mutated genomic germlineDNA is extracted from peripheral blood mononuclear cells (PBMCs). Theapplied NGS approach is confined to the re-sequencing of protein codingregions (exome re-sequencing). For this purpose, exonic DNA from humansamples is captured using vendor-supplied target enrichment kits,followed by sequencing with e.g. a HiSeq2000 (Illumina). Additionally,tumor mRNA is sequenced for direct quantification of gene expression andvalidation that mutated genes are expressed in the patients' tumors. Theresultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized. The de novo identifiedpeptides can then be tested for immunogenicity as described above forthe warehouse, and candidate TUMAPs possessing suitable immunogenicityare selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andoverpresentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of ˜2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from AML cells and since it was determined that thesepeptides are not or at lower levels present in normal tissues, thesepeptides can be used to diagnose the presence of a cancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for AML. Presence of groups of peptides can enableclassification or sub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, and with reference to theaccompanying figures, nevertheless, without being limited thereto. Forthe purposes of the present invention, all references as cited hereinare incorporated by reference in their entireties.

FIGURES

FIGS. 1A through 1D show the over-presentation of various peptides innormal tissues (white bars) and AML (black bars). FIG. 1A, Gene symbol:GNA15, Peptide: LLDSAVYYL (SEQ ID NO.: 1) Tissues from left to right: 5adipose tissues, 5 adrenal glands, 15 blood vessels, 14 brains, 7breasts, 7 esophagi, 2 eyes, 3 gallbladders, 16 hearts, 17 kidneys, 20large intestines, 24 livers, 49 lungs, 7 lymph nodes, 12 nerves, 2ovaries, 8 pancreases, 6 parathyroid glands, 1 peritoneum, 5 pituitaryglands, 7 placentas, 1 pleura, 3 prostates, 7 salivary glands, 5skeletal muscles, 11 skins, 4 small intestines, 12 spleens, 5 stomachs,5 testes, 2 thymi, 2 thyroid glands, 11 tracheas, 7 ureters, 8 urinarybladders, 6 uteri, 24 blood cells, 10 bone marrows, 17 AML samples. Thepeptide has additionally been detected on 1/17 gallbladder and bile ductcancers, 2/18 melanomas, 1/20 ovarian cancers, 2/17 esophageal cancers,4/85 non-small cell lung cancers, 1/15 urinary bladder cancers and 1/16uterine cancers. FIG. 1B Gene symbol: DDX50, Peptide: LLWGDIMEL (SEQ IDNO.: 7) Tissues from left to right: 5 adipose tissues, 5 adrenal glands,15 blood vessels, 14 brains, 7 breasts, 7 esophagi, 2 eyes, 3gallbladders, 16 hearts, 17 kidneys, 20 large intestines, 24 livers, 49lungs, 7 lymph nodes, 12 nerves, 2 ovaries, 8 pancreases, 6 parathyroidglands, 1 peritoneum, 5 pituitary glands, 7 placentas, 1 pleura, 3prostates, 7 salivary glands, 5 skeletal muscles, 11 skins, 4 smallintestines, 12 spleens, 5 stomachs, 5 testes, 2 thymi, 2 thyroid glands,11 tracheas, 7 ureters, 8 urinary bladders, 6 uteri, 24 blood cells, 10bone marrows, 17 AML samples. The peptide has additionally been detectedon 2/20 non-hodgkin lymphomas. FIG. 1C Gene symbols: TMEM183B, TMEM183A,Peptide: VILDPVHSV (SEQ ID NO.: 10) Tissues from left to right: 5adipose tissues, 5 adrenal glands, 15 blood vessels, 14 brains, 7breasts, 7 esophagi, 2 eyes, 3 gallbladders, 16 hearts, 17 kidneys, 20large intestines, 24 livers, 49 lungs, 7 lymph nodes, 12 nerves, 2ovaries, 8 pancreases, 6 parathyroid glands, 1 peritoneum, 5 pituitaryglands, 7 placentas, 1 pleura, 3 prostates, 7 salivary glands, 5skeletal muscles, 11 skins, 4 small intestines, 12 spleens, 5 stomachs,5 testes, 2 thymi, 2 thyroid glands, 11 tracheas, 7 ureters, 8 urinarybladders, 6 uteri, 24 blood cells, 10 bone marrows, 17 AML samples. Thepeptide has additionally been detected on 1/18 breast cancers, 2/20non-hodgkin lymphomas, 1/20 ovarian cancers, 1/85 non-small cell lungcancers, 1/17 small cell lung cancers, 1/16 uterine cancers. FIG. 1DGene symbol: TRIM27, Peptide: ILSDNLRQV (SEQ ID NO.: 138) Samples fromleft to right: 2 cancer cell lines, 1 primary cancer culture, 5 normaltissues (1 adrenal gland, 1 bone marrow, 1 colon, 1 kidney, 1 stomach),34 cancer tissues (2 bone marrow cancers, 2 breast cancers, 1 esophagealcancer, 1 kidney cancer, 3 leukocytic leukemia cancers, 2 liver cancers,8 lung cancers, 5 lymph node cancers, 2 myeloid cells cancers, 1 ovariancancer, 1 prostate cancer, 1 rectum cancer, 2 skin cancers, 3 urinarybladder cancers).

FIGS. 2A through 2C show exemplary expression profiles of source genesof the present invention that are highly over-expressed or exclusivelyexpressed in AML in a panel of normal tissues (white bars) and 11 AMLsamples (black bars). Tissues from left to right: 6 arteries, 2 bloodcells, 2 brains, 1 heart, 2 livers, 3 lungs, 2 veins, 1 adipose tissue,1 adrenal gland, 5 bone marrows, 1 cartilage, 1 colon, 1 esophagus, 2eyes, 2 gallbladders, 1 kidney, 6 lymph nodes, 4 pancreases, 2peripheral nerves, 2 pituitary glands, 1 rectum, 2 salivary glands, 2skeletal muscles, 1 skin, 1 small intestine, 1 spleen, 1 stomach, 1thyroid gland, 7 tracheas, 1 urinary bladder, 1 breast, 5 ovaries, 5placentas, 1 prostate, 1 testis, 1 thymus, 1 uterus, 11 AML samples.FIG. 2A Gene symbol: COL24A1, FIG. 2B Gene symbol: SPNS3, FIG. 2C Genesymbol: KCNE1L.

FIGS. 3A and 3B show exemplary immunogenicity data: flow cytometryresults after peptide-specific multimer staining.

EXAMPLES Example 1

Identification and Quantitation of Tumor Associated Peptides Presentedon the Cell Surface

Tissue Samples

Patients' tumor tissues were obtained from: ProteoGenex Inc. (CulverCity, Calif., USA) Tissue Solutions Ltd (Glasgow, UK); UniversityHospital Bonn (Bonn, Germany); University Hospital Tübingen (Tübingen,Germany).

Normal tissues were obtained from: Asterand (Detroit, Mich., USA &Royston, Herts, UK) Bio-Options Inc. (Brea, Calif., USA); BioServe(Beltsville, Md., USA); Capital BioScience Inc. (Rockville, Md., USA);Geneticist Inc. (Glendale, Calif., USA); Kyoto Prefectural University ofMedicine (KPUM) (Kyoto, Japan); ProteoGenex Inc. (Culver City, Calif.,USA); Tissue Solutions Ltd (Glasgow, UK); University Hospital Geneva(Geneva, Switzerland); University Hospital Heidelberg (Heidelberg,Germany); University Hospital Munich (Munich, Germany); UniversityHospital Tübingen (Tübingen, Germany).

Written informed consents of all patients had been given before surgeryor autopsy. Tissues were shock-frozen immediately after excision andstored until isolation of TUMAPs at −70° C. or below.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk et al., 1991; Seeger et al., 1999) using theHLA-A*02-specific antibody BB7.2, the HLA-A, —B, C-specific antibodyW6/32, CNBr-activated sepharose, acid treatment, and ultrafiltration.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (nanoAcquity UPLCsystem, Waters) and the eluting peptides were analyzed in LTQ-velos andfusion hybrid mass spectrometers (ThermoElectron) equipped with an ESIsource. Peptide pools were loaded directly onto the analyticalfused-silica micro-capillary column (75 μm i.d.×250 mm) packed with 1.7μm C18 reversed-phase material (Waters) applying a flow rate of 400 nLper minute. Subsequently, the peptides were separated using a two-step180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nLper minute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe nanoESI source. The LTQ-Orbitrap mass spectrometers were operated inthe data-dependent mode using a TOPS strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the orbitrap(R=30 000), which was followed by MS/MS scans also in the orbitrap(R=7500) on the 5 most abundant precursor ions with dynamic exclusion ofpreviously selected ions. Tandem mass spectra were interpreted bySEQUEST and additional manual control. The identified peptide sequencewas assured by comparison of the generated natural peptide fragmentationpattern with the fragmentation pattern of a synthetic sequence-identicalreference peptide.

Label-free relative LC-MS quantitation was performed by ion countingi.e. by extraction and analysis of LC-MS features (Mueller et al.,2007). The method assumes that the peptide's LC-MS signal areacorrelates with its abundance in the sample. Extracted features werefurther processed by charge state deconvolution and retention timealignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MSfeatures were cross-referenced with the sequence identification resultsto combine quantitative data of different samples and tissues to peptidepresentation profiles. The quantitative data were normalized in atwo-tier fashion according to central tendency to account for variationwithin technical and biological replicates. Thus each identified peptidecan be associated with quantitative data allowing relativequantification between samples and tissues. In addition, allquantitative data acquired for peptide candidates was inspected manuallyto assure data consistency and to verify the accuracy of the automatedanalysis. For each peptide a presentation profile was calculated showingthe mean sample presentation as well as replicate variations. Theprofiles juxtapose AML samples to a baseline of normal tissue samples.Presentation profiles of exemplary over-presented peptides are shown inFIGS. 1A through 1D. Presentation scores for exemplary peptides areshown in Table 8.

TABLE 8 Presentation scores. The table lists peptidesthat are very highly over-presented on tumorscompared to a panel of normal tissues (+++),highly over-presented on tumors compared to apanel of normal tissues (++) or over-presentedon tumors compared to a panel of normal tissues(+). The panel of normal tissues consideredrelevant for comparison with tumors consistedof: adipose tissue, adrenal gland, artery,blood cells, bone marrow, brain, central nerve,colon, duodenum, esophagus, eye, gallbladder,heart, kidney, liver, lung, lymph node,pancreas, parathyroid gland, peripheral nerve,peritoneum, pituitary gland, pleura, rectum,salivary gland, skeletal muscle, skin, smallintestine, spleen,stomach, thyroid gland,trachea, ureter, urinary bladder, vein.  SEQ ID Peptide No. SequencePresentation   1 LLDSAVYYL +++   2 VLLKAVAQA +++   3 ALYDKTKRIFL +++   4FLPDAFVTM +++   5 FLYYEDLVSC +++   7 LLWGDIMEL +++   8 LLWPGAALLV +++ 10 VILDPVHSV +++  11 ILTQIDHIL +++  12 ALIESNTAL +++  13 ALVPGVTQV +++ 14 ALWWGTITL +++  15 FIDEEVEDMYL +++  16 FLDTQAPSL +++  17 FLLGLSEQL+++  18 GIIEENWQL +++  19 GIVEYLSLV +++  20 GLDAFLLEL +++  21 GLFHGTELL+++  22 GLLQLDTAFV +++  23 GLLQPPVRIV +++  24 GLVELLNRV +++  25GVEGSLIVEKI +++  26 NAGVEGSLIVEKI +++  27 KANPALYVL +++  28 LLDQMETPL+++  29 RLGPSVVGL +++  30 SIISDSSAL +++  31 SLFVFIPMV +++  32 SLSDRSWHL+++  33 TIMNQEKLAKL +++  35 VLFEHAVGYAL +++  36 VLGPSPSSV +++  37VVAPAPVVEAV +++  38 AAIASTPTL +++  39 AIFAGTMQL +++  40 ALAAGGYDVEKN +++ 41 ALFILPFVSV +++  42 ALTTYTIEV +++  43 AMLDFVSSL +++  44 FAVDNVGNRTL+++  45 FLFTDVLLM +++  46 GLDQYLQEV +++  47 GLIJPNVQL +++  48 IAIEALTQL+++  49 IIDDNHAIV +++  50 IIWATSLLL +++  51 SLLSSSLNV +++  52 IVDPVDSTL+++  53 KAFLGELTL +++  54 KLPEFLVQL +++  55 KTLDLINKL +++  56 LANPTTSAL+++  57 LLDFGSLSNLQV +++  58 LLLATLQEA +++  59 LSVPEGAIVSL +++  60NLLNVLEYL +++  61 FLLPGVLLSEA +++  62 RLLFNLSEV +++  63 RLNDTIQLL +++ 64 SLANIKIWV +++  65 SLEEQLSALTL +++  66 SLKNEVGGLV +++  67 SLQDRVIAL+++  68 TGITTPVASV +++  69 TIIGLVRVI +++  70 TLTDSNAQL +++  71 TLTSSLATV+++  72 VAFPSGDASSL +++  73 VAIPDVDPL +++  74 VANPVLYVL +++  75VLAPLGFTL +++  76 VLLJPVPEL +++  77 VLNMKPPEI +++  78 VLSEVECHL +++  79YLMDPDTFTF +++  80 YLTEALQSI +++  81 YVTEELPQL +++  82 LLPDNFIAA ++  84GLLGSVLTI +++  85 GLVPFGLYL +++  86 HLLGDPMANV +  87 ILKPFGNSI +++  88LALNFGSTL +++  89 LLESPVDGWQV +++  90 LLLDTVTSI +++  91 RLAHYIDRV +++ 92 RLWDIQHQL +++  93 SLINDVLAE +++  94 SLLEFAQYL +++  95 SVAEINVLI +++ 96 TLLASYVFL +++  97 TIMTGVIGV ++  98 TQFGFLMEV +++  99 YLAPFSLSNY +++100 AAPAVLGEVDTSLV +++ 101 AINKDPEAPIFQV + 102 ALAQGAERV +++ 103ALGDFGIRL ++ 104 ALIPETTTL ++ 105 GVFALVTAV + 106 ALLEELERSTL + 107ALLGMLPLL +++ 108 ELEMNSDLKAQL +++ 109 GLLAVPLLAA +++ 110 GLTHTAVVPLDLV+++ 111 GVEPAADGKGVVVV +++ 112 ILRDALNQA ++ 113 NLQSEVEGV +++ 114RLAQEAAQV ++ 115 SLPDLTTPL +++ 116 TILEILPEL +++ 117 TILPTILFL ++ 118TLLTVLTQA +++ 119 TLTDELAAL +++ 120 VIQDLVVSV +++ 121 VLQAGQYGV +++ 122VLYLEEVLL +++ 123 YTVKINPTL +++ 124 GLPELVIQL +++ 125 GLFGYLVFL ++ 126GLLPQQIQAV ++ 127 KIISALPQL ++ 128 NLSTKTEAV +++ 129 RMAVLNEQV + 130GVLGNALEGV + 131 SLFSGSLEPV + 132 SLYPVLNFL ++ 133 TVIGTLLFL + 135GIIDRIFQA ++ 136 GLSSIETLL +++ 137 ILAPLAWDL +++ 139 NLIIFSPSV + 141GLLPPLRIPELL ++ 142 GLSDGYGFTT +++ 143 YLLPHILVY +++ 144 GLFMGLVLV + 147FALPILNAL +++ 148 FLYFEDHGL ++ 149 GLAEILVLV + 151 GLLPFPEVTL + 152GLSNHIAAL ++ 153 GLYTGQLAL +++ 154 IIADNIIFL ++ 155 ILDLIQVFV + 157ILTETQQGL +++ 159 LLPLAPAAA ++ 161 SLIGIAIAL + 162 SLLDFLTFA + 163SLMIDLIEV ++ 164 SLNPQEDVEF + 165 SLVDRVAAA +++ 166 VLFPLNLQL + 167VLLDVALGL + 168 VLLFETALL + 169 VLQDPIWLL +++ 170 IVTEVAVGV +++ 171KLLKQVDFL +++ 172 KLLWGDIMEL +++ 173 KMQETLVGL +++ 174 NLTENLQYV +++ 175KMDJFLDMQL +++ 176 HLWTGEEQL +++ 177 KITTVIQHV +++ 178 KLWPLFVKL +++ 179RLISTLENL +++ 180 ALDQEIIEV +++ 181 KLLNHVTQL +++ 183 SVIGVSPAV +++ 184RMTDQEAIQDL +++ 185 RLIPIIVLL +++ 186 IILDEAHNV ++ 187 MLPPPPLTA ++ 188RLLDFPTLL +++

Example 2

Expression Profiling of Genes Encoding the Peptides of the Invention

Over-presentation or specific presentation of a peptide on tumor cellscompared to normal cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in normal tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on normal tissues.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above(see Example 1) after written informed consent had been obtained fromeach patient. Tumor tissue specimens were snap-frozen immediately aftersurgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRI Reagent(Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN,Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues for RNASeq experiments was obtainedfrom: Asterand (Detroit, Mich., USA & Royston, Herts, UK); BioCat GmbH(Heidelberg, Germany); BioServe (Beltsville, Md., USA); CapitalBioScience Inc. (Rockville, Md., USA); Geneticist Inc. (Glendale,Calif., USA); Istituto Nazionale Tumori “Pascale” (Naples, Italy);ProteoGenex Inc. (Culver City, Calif., USA); University HospitalHeidelberg (Heidelberg, Germany)

Total RNA from tumor tissues for RNASeq experiments was obtained from:Asterand (Detroit, Mich., USA & Royston, Herts, UK); ProteoGenex Inc.(Culver City, Calif., USA); Tissue Solutions Ltd (Glasgow, UK);University Hospital Bonn (Bonn, Germany)

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

RNAseq Experiments

Gene expression analysis of—tumor and normal tissue RNA samples wasperformed by next generation sequencing (RNAseq) by CeGaT (Tübingen,Germany). Briefly, sequencing libraries are prepared using the IlluminaHiSeq v4 reagent kit according to the provider's protocol (IlluminaInc., San Diego, Calif., USA), which includes RNA fragmentation, cDNAconversion and addition of sequencing adaptors. Libraries derived frommultiple samples are mixed equimolar and sequenced on the Illumina HiSeq2500 sequencer according to the manufacturer's instructions, generating50 bp single end reads. Processed reads are mapped to the human genome(GRCh38) using the STAR software. Expression data are provided ontranscript level as RPKM (Reads Per Kilobase per Million mapped reads,generated by the software Cufflinks) and on exon level (total reads,generated by the software Bedtools), based on annotations of the ensemblsequence database (Ensembl77). Exon reads are normalized for exon lengthand alignment size to obtain RPKM values. Exemplary expression profilesof source genes of the present invention that are highly over-expressedor exclusively expressed in AML are shown in FIGS. 2A through 2C.Expression scores for further exemplary genes are shown in Table 9.

TABLE 9 Expression scores. The table lists peptides fromgenes that are very highly over-expressed in tumorscompared to a panel of normal tissues (+++), highlyover-expressed in tumors compared to a panel ofnormal tissues (++) or over-expressed in tumorscompared to a panel of normal tissues (+). Thebaseline for this score was calculatedfrom measurements of the following relevant normaltissues: adipose tissue, adrenal gland, artery,blood cells, bone marrow, brain, cartilage, colon,esophagus, eye, gallbladder, heart, kidney, liver,lung, lymph node, pancreas, peripheral nerve,pituitary gland, rectum, salivary gland, skeletalmuscle, skin, small intestine, spleen, stomach,thyroid gland, trachea, urinary bladder, vein. Incase expression data for several samples of thesame tissue type were available, the arithmeticmean of all respective samples was used for the calculation. GeneSEQ ID No Sequence Expression   1 LLDSAVYYL +++   3 ALYDKTKRIFL +++   4FLPDAFVTM +++   5 FLYYEDLVSC +++   8 LLWPGAALLV +  12 ALIESNTAL +++  16FLDTQAPSL +++  17 FLLGLSEQL +  36 VLGPSPSSV ++  41 ALFILPFVSV ++  42ALTTYTIEV +  50 IIWATSLLL ++  51 SLLSSSLNV ++  60 NLLNVLEYL ++  61FLLPGVLLSEA ++  63 RLNDTIQLL ++  64 SLANIKIWV +++  92 RLWDIQHQL +  96TLLASYVFL +++  97 TIMTGVIGV +++ 102 ALAQGAERV +++ 103 ALGDFGIRL +++ 114RLAQEAAQV + 146 ALDTRVVEL +++ 165 SLVDRVAAA ++ 175 KMDJFLDMQL ++ 176HLWTGEEQL ++ 182 SLSEYDQCL +++

Example 3

In Vitro Immunogenicity for MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, the inventors performed investigationsusing an in vitro T-cell priming assay based on repeated stimulations ofCD8+ T cells with artificial antigen presenting cells (aAPCs) loadedwith peptide/MHC complexes and anti-CD28 antibody. This way theinventors could show immunogenicity for HLA-A*0201 restricted TUMAPs ofthe invention, demonstrating that these peptides are T-cell epitopesagainst which CD8+ precursor T cells exist in humans (Table 10).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*02leukapheresis products via positive selection using CD8 microbeads(Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors obtainedfrom the University clinics Mannheim, Germany, after informed consent.

PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium(TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nümberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed in a highly defined in vitro system using fourdifferent pMHC molecules per stimulation condition and 8 different pMHCmolecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidincoated polystyrene particles (Bangs Laboratories, Illinois, USA).

pMHC used for positive and negative control stimulations wereA*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 199) from modifiedMelan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO.200), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3 days at 37° C. Half of the medium was then exchanged by fresh TCMsupplemented with 80 U/ml IL-2 and incubating was continued for 4 daysat 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using eight different pMHCmolecules per condition, a two-dimensional combinatorial coding approachwas used as previously described (Andersen et al., 2012) with minormodifications encompassing coupling to 5 different fluorochromes.Finally, multimeric analyses were performed by staining the cells withLive/dead near IR dye (Invitrogen, Karlsruhe, Germany), CD8-FITCantibody clone SK1 (BD, Heidelberg, Germany) and fluorescent pMHCmultimers. For analysis, a BD LSRII SORP cytometer equipped withappropriate lasers and filters was used. Peptide specific cells werecalculated as percentage of total CD8+ cells. Evaluation of multimericanalysis was done using the FlowJo software (Tree Star, Oregon, USA). Invitro priming of specific multimer+CD8+ lymphocytes was detected bycomparing to negative control stimulations. Immunogenicity for a givenantigen was detected if at least one evaluable in vitro stimulated wellof one healthy donor was found to contain a specific CD8+ T-cell lineafter in vitro stimulation (i.e. this well contained at least 1% ofspecific multimer+ among CD8+ T-cells and the percentage of specificmultimer+ cells was at least 10× the median of the negative controlstimulations).

In Vitro Immunogenicity for AML Peptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated by generation of peptide specific T-cell lines. Exemplaryflow cytometry results after TUMAP-specific multimer staining for 2peptides of the invention are shown in FIGS. 3A and 3B together withcorresponding negative controls. Results for 10 peptides from theinvention are summarized in Table 10.

TABLE 10 in vitro immunogenicity of HLA class I peptides ofthe invention Exemplary results of in vitro immunogenicityexperiments conducted by the applicant for thepeptides of the invention. Seq ID NO: Sequence wells 189 RLFEEVLGV ++190 SLYKGLLSV ++ 191 ALSVLRLAL ++ 192 GLAALAVHL ++ 193 FLLAEDTKV ++ 194LLWGNLPEI ++ 195 FLFVDPELV +++ 196 ILVDWLVQV ++ 197 VLLNEILEQV ++ 198KIQEILTQV ++ <20% = +; 20%-49% = ++; 50%-69% = +++; >= 70% = ++++

Example 4

Synthesis of Peptides

All peptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy. Identity and purity ofeach individual peptide have been determined by mass spectrometry andanalytical RP-HPLC. The peptides were obtained as white to off-whitelyophilizes (trifluoro acetate salt) in purities of >50%. All TUMAPs arepreferably administered as trifluoro-acetate salts or acetate salts,other salt-forms are also possible.

REFERENCE LIST

-   Alcoser, S. Y. et al., BMC. Biotechnol. 11 (2011): 124-   Allison, J. P. et al., Science 270 (1995): 932-933-   Andersen, R. S. et al., Nat. Protoc. 7 (2012): 891-902-   Anderson, N. L. et al., J Proteome. Res 11 (2012): 1868-1878-   Appay, V. et al., Eur. J Immunol. 36 (2006): 1805-1814-   Banchereau, J. et al., Cell 106 (2001): 271-274-   Beatty, G. et al., J Immunol 166 (2001): 2276-2282-   Beggs, J. D., Nature 275 (1978): 104-109-   Benjamini, Y. et al., Journal of the Royal Statistical    Society.Series B (Methodological), Vol. 57 (1995): 289-300-   Boulter, J. M. et al., Protein Eng 16 (2003): 707-711-   Braumuller, H. et al., Nature (2013)-   Brossart, P. et al., Blood 90 (1997): 1594-1599-   Bruckdorfer, T. et al., Curr. Pharm. Biotechnol. 5 (2004): 29-43-   Card, K. F. et al., Cancer Immunol Immunother. 53 (2004): 345-357-   Chanock, S. J. et al., Hum. Immunol. 65 (2004): 1211-1223-   Cohen, C. J. et al., J Mol Recognit. 16 (2003a): 324-332-   Cohen, C. J. et al., J Immunol 170 (2003b): 4349-4361-   Cohen, S. N. et al., Proc. Natl. Acad. Sci. U.S.A 69 (1972):    2110-2114-   Coligan, J. E. et al., Current Protocols in Protein Science (1995)-   Colombetti, S. et al., J Immunol. 176 (2006): 2730-2738-   Dengjel, J. et al., Clin Cancer Res 12 (2006): 4163-4170-   Denkberg, G. et al., J Immunol 171 (2003): 2197-2207-   Falk, K. et al., Nature 351 (1991): 290-296-   Fong, L. et al., Proc. Natl. Acad. Sci. U.S.A 98 (2001): 8809-8814-   Forsey, R. W. et al., Biotechnol. Lett. 31 (2009): 819-823-   Gabrilovich, D. I. et al., Nat Med. 2 (1996): 1096-1103-   Gattinoni, L. et al., Nat Rev. Immunol 6 (2006): 383-393-   Gnjatic, S. et al., Proc Natl. Acad. Sci. U.S.A 100 (2003):    8862-8867-   Godkin, A. et al., Int. Immunol 9 (1997): 905-911-   Green, M. R. et al., Molecular Cloning, A Laboratory Manual 4th    (2012)-   Greenfield, E. A., Antibodies: A Laboratory Manual 2nd (2014)-   Hwang, M. L. et al., J Immunol. 179 (2007): 5829-5838-   Jung, G. et al., Proc Natl Acad Sci USA 84 (1987): 4611-4615-   Kibbe, A. H., Handbook of Pharmaceutical Excipients rd (2000)-   Krieg, A. M., Nat Rev. Drug Discov. 5 (2006): 471-484-   Liddy, N. et al., Nat Med. 18 (2012): 980-987-   Ljunggren, H. G. et al., J Exp. Med. 162 (1985): 1745-1759-   Longenecker, B. M. et al., Ann N. Y. Acad. Sci. 690 (1993): 276-291-   Lonsdale, J., Nat. Genet. 45 (2013): 580-585-   Lukas, T. J. et al., Proc. Natl. Acad. Sci. U.S.A 78 (1981):    2791-2795-   Lundblad, R. L., Chemical Reagents for Protein Modification 3rd    (2004)-   Meziere, C. et al., J Immunol 159 (1997): 3230-3237-   Morgan, R. A. et al., Science 314 (2006): 126-129-   Mori, M. et al., Transplantation 64 (1997): 1017-1027-   Mortara, L. et al., Clin Cancer Res. 12 (2006): 3435-3443-   Mueller, L. N. et al., J Proteome. Res 7 (2008): 51-61-   Mueller, L. N. et al., Proteomics. 7 (2007): 3470-3480-   Mumberg, D. et al., Proc. Natl. Acad. Sci. U.S.A 96 (1999):    8633-8638-   Pinheiro, J. et al., nlme: Linear and Nonlinear Mixed Effects Models    (2015)-   Plebanski, M. et al., Eur. J Immunol 25 (1995): 1783-1787-   Porta, C. et al., Virology 202 (1994): 949-955-   Rammensee, H. G. et al., Immunogenetics 50 (1999): 213-219-   Rini, B. I. et al., Cancer 107 (2006): 67-74-   Rock, K. L. et al., Science 249 (1990): 918-921-   Rodenko, B. et al., Nat Protoc. 1(2006): 1120-1132-   Saiki, R. K. et al., Science 239 (1988): 487-491-   Seeger, F. H. et al., Immunogenetics 49 (1999): 571-576-   Sherman, F. et al., Laboratory Course Manual for Methods in Yeast    Genetics (1986)-   Silva, L. P. et al., Anal. Chem. 85 (2013): 9536-9542-   Singh-Jasuja, H. et al., Cancer Immunol. Immunother. 53 (2004):    187-195-   Small, E. J. et al., J Clin Oncol. 24 (2006): 3089-3094-   Sturm, M. et al., BMC. Bioinformatics. 9 (2008): 163-   Teufel, R. et al., Cell Mol Life Sci. 62 (2005): 1755-1762-   Tran, E. et al., Science 344 (2014): 641-645-   Walter, S. et al., J Immunol 171 (2003): 4974-4978-   Walter, S. et al., Nat Med. 18 (2012): 1254-1261-   Willcox, B. E. et al., Protein Sci. 8 (1999): 2418-2423-   Zaremba, S. et al., Cancer Res. 57 (1997): 4570-4577

The invention claimed is:
 1. A method of eliciting an immune response ina patient who has cancer, comprising administering to the patient apopulation of activated T cells that kill cancer cells that present apeptide consisting of the amino acid sequence SEQ ID NO: 52, wherein theactivated T cells are cytotoxic T cells produced by in vitro contactingT cells with an antigen presenting cell that expresses the peptide in acomplex with an WIC class I molecule on the surface of the antigenpresenting cell, for a period of time sufficient to activate said Tcell, wherein said cancer is selected from the group consisting of acutemyelogenous leukemia/acute myeloid leukemia (AML) and melanoma.
 2. Themethod of claim 1, wherein the T cells are autologous to the patient. 3.The method of claim 1, wherein the T cells are obtained from a healthydonor.
 4. The method of claim 1, wherein the T cells are obtained fromtumor infiltrating lymphocytes or peripheral blood mononuclear cells. 5.The method of claim 1, wherein the activated T cells are expanded invitro.
 6. The method of claim 1, wherein the peptide presented by thecancer cell is in a complex with an MHC molecule.
 7. The method of claim6, wherein the MHC molecule is a class I MHC molecule.
 8. The method ofclaim 1, wherein the antigen presenting cell is infected withrecombinant virus expressing the peptide.
 9. The method of claim 8,wherein the antigen presenting cell is a dendritic cell or a macrophage.10. The method of claim 1, wherein the population of activated T cellsare administered in the form of a composition.
 11. The method of claim10, wherein the composition further comprises an adjuvant.
 12. Themethod of claim 11, wherein the adjuvant is at least one selected fromCD40 antibody, imiquimod, resiquimod, GM-CSF, cyclophosphamide,sunitinib, bevacizumab, interferon-alpha, interferon-beta, CpGoligonucleotides, poly-(I:C), RNA, sildenafil, and particulateformations with poly(lactide co-glycolide) (PLG), virosomes, interleukin(IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.
 13. Themethod of claim 11, wherein the adjuvant comprises IL-2.
 14. The methodof claim 11, wherein the adjuvant comprises IL-7.
 15. The method ofclaim 11, wherein the adjuvant comprises IL-15.
 16. The method of claim11, wherein the adjuvant comprises IL-21.
 17. The method of claim 11,wherein the adjuvant comprises cyclophosphamide.
 18. The method of claim1, wherein the immune response comprises a cytotoxic T cell response.19. The method of claim 1, wherein the cancer is AML.
 20. The method ofclaim 1, wherein the cancer is melanoma.